**4. Current protocols for stem cell-based therapeutics**

#### **4.1. Mesenchymal stem cells (MSCs)**

MSCs do not express major histocompatibility complex class I or II, permitting adoptive transfer between hosts without triggering acute rejection. In 2006, the International Society for Cellular Therapy (ISCT) established minimal criteria to define human MSCs as follows: MSC must be plastic-adherent when maintained in standard culture conditions; MSC must express CD105, CD73, and CD90 and lack expression of CD45, CD34, CD14 or CD11b, CD79α or CD19, and HLA-DR surface molecules; and MSC must differentiate to osteoblasts, adipocytes, and chondroblasts *in vitro* [63, 64]. The latter position paper adds to the original characterization criteria viability and proliferation features [64]. Cells that fulfill these criteria can be isolated from different sources (such as fat, bone marrow, umbilical cord blood, dental pulp, etc.), but tissue source, donor's age, extent, and conditions of *in vitro* expansion, among others, are known to influence the regenerative potential based on engraftment, paracrine effects, and differentiation capacity of these cells [65]. Freeze-thawing effects on the whole genome expression profile of MSC have been observed, although they did not exceed interdonor differences [66]. This high inherent heterogeneity of MSCs remains a challenge for data 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 adherence to plastic and extended survival *in vitro* [70, 71].

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 func-

tioning. Preclinical safety studies, however, should precede CT in ME/CFS.

MSCs do not express major histocompatibility complex class I or II, permitting adoptive transfer between hosts without triggering acute rejection. In 2006, the International Society for Cellular Therapy (ISCT) established minimal criteria to define human MSCs as follows: MSC must be plastic-adherent when maintained in standard culture conditions; MSC must express CD105, CD73, and CD90 and lack expression of CD45, CD34, CD14 or CD11b, CD79α or CD19, and HLA-DR surface molecules; and MSC must differentiate to osteoblasts, adipocytes, and chondroblasts *in vitro* [63, 64]. The latter position paper adds to the original characterization criteria viability and proliferation features [64]. Cells that fulfill these criteria can be isolated from different sources (such as fat, bone marrow, umbilical cord blood, dental pulp, etc.), but tissue source, donor's age, extent, and conditions of *in vitro* expansion, among others, are known to influence the regenerative potential based on engraftment, paracrine effects, and differentiation capacity of these cells [65]. Freeze-thawing effects on the whole genome expression profile of MSC have been observed, although they did not exceed interdonor differences [66]. This high inherent heterogeneity of MSCs remains a challenge for data

**4. Current protocols for stem cell-based therapeutics**

**4.1. Mesenchymal stem cells (MSCs)**

116 Cell Culture

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

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

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

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

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


Source: Adapted from Jossen et al. [67]. ASC human adipose tissue-derived stromal/stem cells, BM-MSC human bone marrow-derived mesenchymal stem cells, and UCB-MSC umbilical cord-derived mesenchymal stem cells.

survival and regenerative potential of MSCs and also confers resistance to hostile environments associated with inflammation which induces cell death by oxidative stress [88–90],

**Figure 3.** Main operations required to manufacture clinical-grade human MSCs for allogeneic (A) or autologous (B) therapies. USP operations typically include manufacturing of the MCB and WCB, seed cell production, and expansion at a large scale. DSP steps include cell harvest, cell detachment and separation, washing, concentration procedures, and medium exchange. Formulation, fill and finish, and storage and distribution will complete processes prior to clinical

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Chemically defined, xeno-free medium does not involve donor or batch-to-batch variation; neither requires pathogenic agent screenings and presents minimal immunogenicity [91]. Drawbacks are the high cost of commercial versions and the fact that cells grown under these conditions lose their ability to adhere to plastic, requiring additional coating agent steps [92, 93]. Cells grown in xeno-free protocols also show improved proliferation potential when compared with FBS-based growing media with the previously mentioned consequent advantages [76, 92–94]. In addition to the choice of source of proteins and growth factors, basal media, seeding density, oxygen tension, confluency, and dissociation protocols may also influence outcomes. Further studies that carefully control growing conditions and at the time explore aspects such as senescence, genetic stability, immunogenicity and cytokine

*In vivo* ASCs reside under low oxygen tension (physioxia); Chen et al. have recently shown

concentration used in cell culture (20–21%) leads to increased proliferation, migration, and angiogenesis plus decreased senescence and apoptosis suggesting that the maintenance of

It is clear that tissue source and optimal growing conditions for MSC manufacturing will depend on the needs of downstream applications. In this sense it is important to mention that Okolicsanyi et al. have shown that MSCs isolated from the bone marrow of normal donors

native bioactivities may translate into production of superior cell products [95].

) instead of the typical atmospheric oxygen

evidencing the presence of GFs as a conditioning advantage for stem cell production.

production, and transcriptome and proteome are needed.

that the simulation of physioxic conditions (2% O<sup>2</sup>

administration. Source: Jossen et al. [67].

**Table 1.** MSC-based products with marketing authorization for allogeneic and autologous therapies

production to few doses. The allogeneic alternative permits larger cell production by pooling samples from different donors but requires pathological agent screenings. Human derivatives show improved proliferation when compared to FBS-supplemented media reducing the time for cell expansion and lowering threats of senescence and transformation; however, human serum seems to limit osteogenic differentiation [63, 64, 80], and human platelet-poor plasma limits chondrogenesis [81, 82], while human platelet-rich plasma or platelet lysate preserves trilineage differentiation [83–85]. In addition, human platelet lysate, obtained by temperature-shock protocols (freezing platelets from −30 to −80°C during 24 h followed by a thawing a centrifugation step), can be prepared from banked blood with 4 or 5 days passed expiration date [86], making platelet lysate a preferable choice. To avoid MSC senescence, forced expression of telomerase reverse transcriptase (TERT) has been tried [86, 87]; however, nongenetic manipulations will be more suitable for clinical translation. On another side, the serine/threonine kinase AKT activation by plasma rich in growth factors leads to enhanced Culturing Adult Stem Cells for Cell-Based Therapeutics: Neuroimmune Applications http://dx.doi.org/10.5772/intechopen.80714 119

**Figure 3.** Main operations required to manufacture clinical-grade human MSCs for allogeneic (A) or autologous (B) therapies. USP operations typically include manufacturing of the MCB and WCB, seed cell production, and expansion at a large scale. DSP steps include cell harvest, cell detachment and separation, washing, concentration procedures, and medium exchange. Formulation, fill and finish, and storage and distribution will complete processes prior to clinical administration. Source: Jossen et al. [67].

survival and regenerative potential of MSCs and also confers resistance to hostile environments associated with inflammation which induces cell death by oxidative stress [88–90], evidencing the presence of GFs as a conditioning advantage for stem cell production.

Chemically defined, xeno-free medium does not involve donor or batch-to-batch variation; neither requires pathogenic agent screenings and presents minimal immunogenicity [91]. Drawbacks are the high cost of commercial versions and the fact that cells grown under these conditions lose their ability to adhere to plastic, requiring additional coating agent steps [92, 93]. Cells grown in xeno-free protocols also show improved proliferation potential when compared with FBS-based growing media with the previously mentioned consequent advantages [76, 92–94]. In addition to the choice of source of proteins and growth factors, basal media, seeding density, oxygen tension, confluency, and dissociation protocols may also influence outcomes. Further studies that carefully control growing conditions and at the time explore aspects such as senescence, genetic stability, immunogenicity and cytokine production, and transcriptome and proteome are needed.

production to few doses. The allogeneic alternative permits larger cell production by pooling samples from different donors but requires pathological agent screenings. Human derivatives show improved proliferation when compared to FBS-supplemented media reducing the time for cell expansion and lowering threats of senescence and transformation; however, human serum seems to limit osteogenic differentiation [63, 64, 80], and human platelet-poor plasma limits chondrogenesis [81, 82], while human platelet-rich plasma or platelet lysate preserves trilineage differentiation [83–85]. In addition, human platelet lysate, obtained by temperature-shock protocols (freezing platelets from −30 to −80°C during 24 h followed by a thawing a centrifugation step), can be prepared from banked blood with 4 or 5 days passed expiration date [86], making platelet lysate a preferable choice. To avoid MSC senescence, forced expression of telomerase reverse transcriptase (TERT) has been tried [86, 87]; however, nongenetic manipulations will be more suitable for clinical translation. On another side, the serine/threonine kinase AKT activation by plasma rich in growth factors leads to enhanced

Source: Adapted from Jossen et al. [67]. ASC human adipose tissue-derived stromal/stem cells, BM-MSC human bone

**Medicinal product Company hMSC type Indication Marketing authorization** Allostem AlloSource Allogeneic ASC Bone regeneration US medical device

Osteoarthritis Korea

Graft-versus-host

Graft-versus-host

Acute myocardial infarction

subcutaneous adipose

Bone regeneration Korea

disease

disease

tissue

Spinal bone regeneration

Soft tissue defects US medical device

Bone regeneration US medical device

Bone regeneration US medical device

Bone regeneration US medical device

Japan

Korea

Korea

Canada and New Zealand

US medical device

UCB-MSC

BM-MSC

BM-MSC

BM-MSC

BM-MSC

BM-MSC

BM-MSC

BM-MSC

BM-MSC

Cupistem Anterogen Autologous ASC Crohn's fistula Korea

BM-MSC

**Table 1.** MSC-based products with marketing authorization for allogeneic and autologous therapies

marrow-derived mesenchymal stem cells, and UCB-MSC umbilical cord-derived mesenchymal stem cells.

QueenCell Anterogen Autologous ASC Regeneration of

Cartistem Medipost Allogeneic

118 Cell Culture

Grafix Osiris Therapeutics Allogeneic

Prochymal Mesoblast Allogeneic

OsteoCel NuVasive Allogeneic

OvationOS Osiris Therapeutics Allogeneic

TEMCELL HS JCR Pharmaceuticals Allogeneic

Trinity Evolution Orthofix Allogeneic

Trinity Elite Orthofix Allogeneic

Hearticellgram-AMI Pharmicell Autologous

Ossron RMS Autologous

*In vivo* ASCs reside under low oxygen tension (physioxia); Chen et al. have recently shown that the simulation of physioxic conditions (2% O<sup>2</sup> ) instead of the typical atmospheric oxygen concentration used in cell culture (20–21%) leads to increased proliferation, migration, and angiogenesis plus decreased senescence and apoptosis suggesting that the maintenance of native bioactivities may translate into production of superior cell products [95].

It is clear that tissue source and optimal growing conditions for MSC manufacturing will depend on the needs of downstream applications. In this sense it is important to mention that Okolicsanyi et al. have shown that MSCs isolated from the bone marrow of normal donors (from Lonza, Australia), expanded as monolayer cultures, retain multilineage differentiation capacity, including neural marker expression, after 43 days of *in vitro* expansion in the commercial synthetically defined human mesenchymal stem cell-growth media MSCGM-CD™ (Lonza, Australia) [96]. Therefore, these cells could in principle be a source for cell-based therapies of the nervous system.

The regenerative capacity of MSCs has been attributed to their anti-inflammatory immunoregulatory properties. Depending on the milieu composition, MSCs, in fact, exhibit anti- or pro-inflammatory properties (see **Figure 4**) [42, 97–100].

In an early stage of trauma or microbial invasion, when concentration of pro-inflammatory cytokines is low, MSCs present with antimicrobial pro-inflammatory properties of neutrophils [97–102]. As inflammation proceeds and pro-inflammatory cytokines build up, MSCs switch to an anti-inflammatory phenotype. Some of these anti-inflammatory actions include inhibition of anti-inflammatory activities of T cells, natural killer cells, and B cells; skewing macrophages to an M2 immunosuppressive state and monocyte-derived dendritic cells to a regulatory phenotype; and increasing their phagocytic capacity and inhibit mast cell degranulation [103, 104]. Increased immunomodulatory capacity of MSCs correlates with high levels of activated complement C3 [105, 106]. As MSCs express the complement factor H and the complement regulatory protein CD59, MSCs are protected from lysis. All this endows MSCs with the potential to suppress uncontrolled immune responses making them a suitable candidate for inflammation and immune dysfunction therapeutics by themselves or in combination with other cell types.

## **4.2. Induced pluripotent stem cells (iPSCs)**

NSPCs may differentiate into neural cells after transplantation into an injured spinal cord, replacing lost or damaged cells, providing trophic support, restoring connectivity, and facilitating regeneration as a large number of studies have reported [107]. NSPC has produced some degree of functional recovery. The fetal, adult brain and adult spinal cord are the main sources for NSPCs resulting in advantageous cells for transplantation because they can be expanded and self-renewed in culture. Fetal NSPCs can be expanded for long periods by *in vitro* conditions, while adult NSPCs have more limited capabilities.

All the previous reports support the potential use of iPSC-derived NSPCs in SCI. They have significant advantages, such as the lack of ethical controversy regarding their source and the potential for providing autologous transplants, thus avoiding the risk of rejection or side effects associated with immunosuppression. Recent data demonstrated the effect of the microenvironment of the injured spinal cord in the grafted iPSC-derived NSPCs. This pro-inflammatory environment induced proliferation of grafted cells [115]. Therefore, new approaches are needed to promote and guide cell differentiation, as well as to reduce tumorigenicity. Protocols

**Figure 4.** Immunomodulatory action of activated MSCs. Notes: Red arrow, stimulation; black arrow, suppression; bluntended arrow, direct inhibition. Abbreviations: iDC, immature dendritic cell; IL, interleukin; HGF, hepatocyte growth factor; TGF-β, transforming growth factor-β; PGE-2, prostaglandin E2; IDO, indoleamine 2,3-dioxygenase; NO, nitric oxide; PD-L1, programmed death-ligand 1; hMSC, human mesenchymal stem cell; Treg, T regulatory; Th, T helper; CTL, cytotoxic T cell; mDC, mature dendritic cell; PD-1, programmed cell death protein 1; PMN, polymorphonuclear

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The most current iPSC protocols for neural differentiation require GFs or embryoid body formation, decreasing yields and limiting medical applications. Our lab recently developed a simple animal-free medium formula based on the inclusion of insulin and human extracellular matrix components leading to direct conversion of >98% of iPSCs into expandable and functional neural progenitors with neural rosette characteristics [111]. Further differentiation into dopaminergic and spinal motoneurons as well as oligodendrocytes and astrocytes supports the proposal that these neural progenitors retain responsiveness to environmental cues supporting applicability of the protocol for the treatment of neurodegenerative diseases. The fact that this protocol avoids embryoid body formation makes it suitable for the clinic [111]. Formerly, a feeder-free, single-step, and quick (less than 40 days) generation of mature neurons from iPSC strategy using the chemically defined medium mTeSR from STEMCELL

for NSPC reprogrammed cells are actually improved to avoid rejection [116].

leukocyte; NK, NK cell. Source: Zachar et al. [97].

Despite a large number of studies using NSPCs, reviewed by Mothe et al. [108], some important issues such as isolation from their natural niche and their purification and expansion have to be taken in consideration [109]. Also, NSPCs have been reported to promote neuropathic pain, a concerning adverse effect. Most experimental SCI studies with NSPC transplants have involved rodent NSPCs because human NSPCs were either not available or difficult to grow. Human NSPCs have been isolated from the fetal brain and spinal cord of aborted fetuses [110] and postmortem tissue, but actually NSPCs can also be derived from human iPSCs [111].

Human iPSC-derived NSPCs have been transplanted into SCI models [112–114]. In these studies, nonobese diabetic (NOD)-severe combined immunodeficient (SCID) mice were used for SCI. The studies revealed an improved functional recovery with expression of neurotrophic factors from the grafted cells, axonal growth and stimulation of angiogenesis, increased myelination, and new forming synaptic connections between grafted cells and host neurons. In addition these studies showed the safety of human iPSC-derived NSPCs. All studies were performed in the subacute stage with just epicenter transplants.

Culturing Adult Stem Cells for Cell-Based Therapeutics: Neuroimmune Applications http://dx.doi.org/10.5772/intechopen.80714 121

(from Lonza, Australia), expanded as monolayer cultures, retain multilineage differentiation capacity, including neural marker expression, after 43 days of *in vitro* expansion in the commercial synthetically defined human mesenchymal stem cell-growth media MSCGM-CD™ (Lonza, Australia) [96]. Therefore, these cells could in principle be a source for cell-based

The regenerative capacity of MSCs has been attributed to their anti-inflammatory immunoregulatory properties. Depending on the milieu composition, MSCs, in fact, exhibit anti- or

In an early stage of trauma or microbial invasion, when concentration of pro-inflammatory cytokines is low, MSCs present with antimicrobial pro-inflammatory properties of neutrophils [97–102]. As inflammation proceeds and pro-inflammatory cytokines build up, MSCs switch to an anti-inflammatory phenotype. Some of these anti-inflammatory actions include inhibition of anti-inflammatory activities of T cells, natural killer cells, and B cells; skewing macrophages to an M2 immunosuppressive state and monocyte-derived dendritic cells to a regulatory phenotype; and increasing their phagocytic capacity and inhibit mast cell degranulation [103, 104]. Increased immunomodulatory capacity of MSCs correlates with high levels of activated complement C3 [105, 106]. As MSCs express the complement factor H and the complement regulatory protein CD59, MSCs are protected from lysis. All this endows MSCs with the potential to suppress uncontrolled immune responses making them a suitable candidate for inflammation and immune dysfunction therapeutics by themselves or in combination with other cell types.

NSPCs may differentiate into neural cells after transplantation into an injured spinal cord, replacing lost or damaged cells, providing trophic support, restoring connectivity, and facilitating regeneration as a large number of studies have reported [107]. NSPC has produced some degree of functional recovery. The fetal, adult brain and adult spinal cord are the main sources for NSPCs resulting in advantageous cells for transplantation because they can be expanded and self-renewed in culture. Fetal NSPCs can be expanded for long periods by

Despite a large number of studies using NSPCs, reviewed by Mothe et al. [108], some important issues such as isolation from their natural niche and their purification and expansion have to be taken in consideration [109]. Also, NSPCs have been reported to promote neuropathic pain, a concerning adverse effect. Most experimental SCI studies with NSPC transplants have involved rodent NSPCs because human NSPCs were either not available or difficult to grow. Human NSPCs have been isolated from the fetal brain and spinal cord of aborted fetuses [110] and postmortem tissue, but actually NSPCs can also be derived from human iPSCs [111].

Human iPSC-derived NSPCs have been transplanted into SCI models [112–114]. In these studies, nonobese diabetic (NOD)-severe combined immunodeficient (SCID) mice were used for SCI. The studies revealed an improved functional recovery with expression of neurotrophic factors from the grafted cells, axonal growth and stimulation of angiogenesis, increased myelination, and new forming synaptic connections between grafted cells and host neurons. In addition these studies showed the safety of human iPSC-derived NSPCs. All studies were

*in vitro* conditions, while adult NSPCs have more limited capabilities.

performed in the subacute stage with just epicenter transplants.

therapies of the nervous system.

120 Cell Culture

pro-inflammatory properties (see **Figure 4**) [42, 97–100].

**4.2. Induced pluripotent stem cells (iPSCs)**

**Figure 4.** Immunomodulatory action of activated MSCs. Notes: Red arrow, stimulation; black arrow, suppression; bluntended arrow, direct inhibition. Abbreviations: iDC, immature dendritic cell; IL, interleukin; HGF, hepatocyte growth factor; TGF-β, transforming growth factor-β; PGE-2, prostaglandin E2; IDO, indoleamine 2,3-dioxygenase; NO, nitric oxide; PD-L1, programmed death-ligand 1; hMSC, human mesenchymal stem cell; Treg, T regulatory; Th, T helper; CTL, cytotoxic T cell; mDC, mature dendritic cell; PD-1, programmed cell death protein 1; PMN, polymorphonuclear leukocyte; NK, NK cell. Source: Zachar et al. [97].

All the previous reports support the potential use of iPSC-derived NSPCs in SCI. They have significant advantages, such as the lack of ethical controversy regarding their source and the potential for providing autologous transplants, thus avoiding the risk of rejection or side effects associated with immunosuppression. Recent data demonstrated the effect of the microenvironment of the injured spinal cord in the grafted iPSC-derived NSPCs. This pro-inflammatory environment induced proliferation of grafted cells [115]. Therefore, new approaches are needed to promote and guide cell differentiation, as well as to reduce tumorigenicity. Protocols for NSPC reprogrammed cells are actually improved to avoid rejection [116].

The most current iPSC protocols for neural differentiation require GFs or embryoid body formation, decreasing yields and limiting medical applications. Our lab recently developed a simple animal-free medium formula based on the inclusion of insulin and human extracellular matrix components leading to direct conversion of >98% of iPSCs into expandable and functional neural progenitors with neural rosette characteristics [111]. Further differentiation into dopaminergic and spinal motoneurons as well as oligodendrocytes and astrocytes supports the proposal that these neural progenitors retain responsiveness to environmental cues supporting applicability of the protocol for the treatment of neurodegenerative diseases. The fact that this protocol avoids embryoid body formation makes it suitable for the clinic [111].

Formerly, a feeder-free, single-step, and quick (less than 40 days) generation of mature neurons from iPSC strategy using the chemically defined medium mTeSR from STEMCELL 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 and recommend the method to model human pathologies [117].

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

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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

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

controlled and tailored [96, 130].

turnover for potential remyelination [131, 132].

CNS-homing ligand genes along with overexpression of IL-10 [136].

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

**4.4. Extracellular vesicle-based therapeutics**

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 improvements are available [125–127].

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 as part of combinatorial treatments.
