Open access peer-reviewed chapter

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

Written By

Victoria Moreno-Manzano and Elisa Oltra García

Submitted: April 20th, 2018 Reviewed: August 5th, 2018 Published: November 5th, 2018

DOI: 10.5772/intechopen.80714

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Abstract

Pluripotent stem cells can be successfully isolated from a variety of tissues from adult organisms. This fact opens the exciting possibility of cell-based therapies for a large number of clinical treatments. However, the development of optimized protocols to obtain, grow, and cryopreserve cells, as well as that of effective clinical treatment procedures, is no easy task. The therapeutic potential of cells expanded in vitro depends on a multitude of factors including isolation procedures, donor and tissue types, expansion and preservation methods, etc. Researchers are investing great efforts to determine which of these many variables significantly impact downstream performance of in vitro expanded stem cells by studying associated changes in molecular profiles and their effect on the host immune system. This chapter reviews the current status of stem cell production and its derivatives, which are paving the way to different treatments in the clinic. Due to the research interests of our labs, particular emphasis is placed on the potential benefits of stem cell-based therapeutics for the treatment of spinal cord injuries and the neuroimmune disease myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) not only derived from differentiation and cell engraftment mechanisms but also due to the anti-inflammatory and immunoregulatory capacities of these cells.

Keywords

  • mesenchymal stem cell (MSC)
  • induced pluripotent stem cell (iPSC)
  • spinal cord injury (SCI)
  • myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS)
  • extracellular vesicle (EV)

1. Introduction

Stem cells present particular characteristics that make them different from other cell types. Firstly, they are unspecialized self-renewing tissue resident cells, and secondly they can be induced to differentiate into a milieu of specialized cell types, thus holding promise for regenerative medicine. When these cells are isolated from adult fully differentiated tissues, they receive the attribute of adult stem cells, even though they are also present in infants and fetus. Therefore, it would be more appropriate to refer to them as tissue stem cells or mesenchymal stem cells to differentiate them from resident progenitors with limited differentiation capacity. MSCs can be isolated from a large number of tissues, such as bone marrow, adipose tissue, dental pulp, hair follicles, amniotic fluid, Wharton’s jelly in the umbilical cord, and even from nervous or cardiac tissue. MSCs are multipotent and can be differentiated into chondrocytes, adipocytes, and osteoblasts under proper conditions [1, 2]. MSCs can be cloned and expanded in vitromore than a million fold without losing their differentiation potential [3] constituting, theoretically, a rich resource for tissue repair. However, their sensitivity to environmental cues and genetic factors together with a lack of standardized good manufacturing procedures (GMPs) using defined components has hampered their true therapeutic potential. Since the finding by Bartholomew et al. that MSCs inhibit mixed lymphocyte reactions and prevent the rejection of allogeneic skin grafts [4], a large number of reports have evidenced that MSCs are immunosuppressive and immunoregulatory, properties that can be harnessed therapeutically. However, challenges to fully understand and control MSC regenerative potential remain.

In addition to MSC, the reprograming of terminally differentiated cells or induction of de-differentiation by the introduction of particular sets of transcription factors [5, 6, 7] opened an additional avenue of opportunities in the field of regenerative medicine. iPScs or induced pluripotent stem cells facilitate the production of patient-specific cells overcoming immune rejection and also ethical concerns. Although they have shown their value in the generation of in vitromodels of human disease [8, 9], the low efficiency of reprogramming events and the safety concerns associated with the process of reprogramming has prevented their use in the clinic [6, 10].

Based on the research interests of our labs, this chapter, while reviewing the advances to generate clinical-grade stem cells or their by-products, highlights the potential benefits of stem cell-based therapeutics for the treatment of spinal cord injuries (SCI) and the neuroimmune disease myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS).

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2. Stem cell therapy for spinal cord repair

2.1. Spinal cord injury pathological events and timing sequence

Spinal cord injury (SCI) is often mentioned among the first conditions for which stem cells may provide a new therapy. While recent decades have brought significant improvements in rescuing neuronal activity after SCI at preclinical phases testing several individual approaches, translation to the clinic still remains inefficiently explored. Management for SCI efficient treatment is a difficult task by the intrinsic nature of the pathological cascade of events that makes the SCI a dynamic and progressive disorder. The sentence “Time is spine” defines the crucial importance of timing to rapidly diagnose patients and implement neuroprotective interventions during the acute injury phase (≤2 h) in order to diminish the devastating effects of the secondary phase of the injury (≥2–48 h) which are known to be key determinants of the final extent of neurological deficits. The secondary injury leads to necrosis and/or apoptosis of neurons and glial cells, such as oligodendrocytes, which can lead to demyelination and the loss of neural circuits. Later, in a subacute phase (2–4 days after injury), further ischemia occurs owing to ongoing edema, vessel thrombosis, and vasospasm. Persistent inflammatory cell infiltration causes further cell death and formation of very toxic cystic microcavities over time. Astrocytes, fibroblast, and pericytes proliferate and deposit extracellular matrix molecules into the perilesional area in the already intermediate and chronic phases, few weeks after SCI, when axons continue degenerating (Figure 1a) [11].

Figure 1.

Summary of physiopathological events after SCI (a) and stem cell transplantation (b).

Spontaneous regeneration during and after having reached the chronic stage occurs due to the neuroplasticity capacity of the central nervous system (CNS); however, very limited gain of function is obtained decreasing advancing age, attributed to both extrinsic and intrinsic factors that modulate further onset, severity, and progression of the injury [12]. The cumulative myelin-associated protein anchorage to myelin sheet debris, in and around the epicenter of the injury, has a strong inhibitory nature. Nogo-A (reticulon-4 isoform A) and myelin-associated glycoprotein (MAG), among other myelin-associated proteins, bind to NOGO receptors to activate the GTPase Rho A, which activates Rho-associated protein kinase (ROCK), a regulator of further downstream effectors, leading to apoptosis and growth-cone collapse of regenerating axons involving neurite retraction [13, 14, 15, 16, 17]. Additional external barriers are potently adding to the inhibition of regeneration like the hypertrophic astrocytes and the reactive chemical scar with a number of axonal growth inhibitory chondroitin sulfate proteoglycans (CSPGs) [18].

The chronic SCI repair demands an intensive effort to overcome the impediments and enhance the intrinsic axon regeneration involving an efficient anatomical reorganization [19, 20]. Fortunately, although long distances for axonal reconnection or spared degenerated tracts are normally required, involving a long-term process (a rate of 1 mm/month for axon growth is estimated), it has been shown that as little as 10% of particular tracts can subserve substantial function [19, 20]. This in fact allows hypothesizing for a real recovery, mediated by bridging and partially reconnecting the spared axons allowing subsequent plasticity. Additionally, both, humans and rats, can regain a degree of function after incomplete injury, thought to be mostly due to local structural rearrangements, such as collateral sprouting from remaining axons in the gray matter, rather than by long-distance regeneration of axons in the white matter (Figure 1b) [21].

2.2. Stem cell therapy for SCI repair

Cell transplantation methods constitute a very promising strategy for SCI repair. Numerous studies with a diversity of cell types have clearly showed benefits to different extents, for instance, therapeutic effects in sensorimotor function recovery in preclinical models [22, 23] and in several ongoing clinical trials [24, 25, 26].

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

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 outcomes after SCI [35].

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 vitrodifferentiated 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 evaluated.

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

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 anti-inflammatory properties [42, 43, 44, 45]. As reviewed by Wang et al., as of April 2016, over 500 MSC-related 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].

Figure 2.

Summary of the number of clinical trials using MSC therapy in immune- or inflammation-mediated diseases, as registered on the websitehttps://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].

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, 43, 44, 45, 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, 47, 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, 50, 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, 50, 51].

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 cell-based 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.

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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 vitroexpansion, 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, 68, 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].

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].

Medicinal productCompanyhMSC typeIndicationMarketing authorization
AllostemAlloSourceAllogeneic ASCBone regenerationUS medical device
CartistemMedipostAllogeneic UCB-MSCOsteoarthritisKorea
GrafixOsiris TherapeuticsAllogeneic BM-MSCSoft tissue defectsUS medical device
ProchymalMesoblastAllogeneic BM-MSCGraft-versus-host diseaseCanada and New Zealand
OsteoCelNuVasiveAllogeneic BM-MSCSpinal bone regenerationUS medical device
OvationOSOsiris TherapeuticsAllogeneic BM-MSCBone regenerationUS medical device
TEMCELL HSJCR PharmaceuticalsAllogeneic BM-MSCGraft-versus-host diseaseJapan
Trinity EvolutionOrthofixAllogeneic BM-MSCBone regenerationUS medical device
Trinity EliteOrthofixAllogeneic BM-MSCBone regenerationUS medical device
Hearticellgram-AMIPharmicellAutologous BM-MSCAcute myocardial infarctionKorea
CupistemAnterogenAutologous ASCCrohn’s fistulaKorea
QueenCellAnterogenAutologous ASCRegeneration of subcutaneous adipose tissueKorea
OssronRMSAutologous BM-MSCBone regenerationKorea

Table 1.

MSC-based products with marketing authorization for allogeneic and autologous therapies

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.

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.

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].

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, 78, 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 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, 84, 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 survival and regenerative potential of MSCs and also confers resistance to hostile environments associated with inflammation which induces cell death by oxidative stress [88, 89, 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, 93, 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.

In vivoASCs reside under low oxygen tension (physioxia); Chen et al. have recently shown that the simulation of physioxic conditions (2% O2) 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 vitroexpansion 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, 98, 99, 100].

Figure 4.

Immunomodulatory action of activated MSCs. Notes: Red arrow, stimulation; black arrow, suppression; blunt-ended 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].

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, 98, 99, 100, 101, 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 vitroconditions, while adult NSPCs have more limited capabilities.

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, 113, 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.

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].

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, 123, 124]. Excellent reviews describing more details for reprogramming protocol improvements are available [125, 126, 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 vitrowithout senescing. Thus, SCI stem cell-based therapeutics may benefit including PSCs as part of combinatorial treatments.

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].

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, 43, 44, 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.

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 TNF-α) [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 steroid-refractory 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 long-term 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.

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5. Conclusions

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 vitroexpanded 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 vivoproperties of immortal cell types and the use of genetically manipulated cells raise regulation hurdles for their use in the clinic.

Preconditioning of in vitroexpanded 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.

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Acknowledgments

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).

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Conflict of interest

Authors declare no conflict of interest.

References

  1. 1. Uccelli A, Moretta L, Pistoia V. Mesenchymal stem cells in health and disease. Nature Reviews. Immunology. 2008;8(9):726-736. DOI: 10.1038/nri2395
  2. 2. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284(5411):143-147
  3. 3. Huss R. Isolation of primary and immortalized CD34-hematopoietic and mesenchymal stem cells from various sources. Stem Cells. 2000;18(1):1-9. Review
  4. 4. Bartholomew A, Sturgeon C, Siatskas M, Ferrer K, McIntosh K, Patil S, et al. Mesenchymal stem cells suppress lymphocyte proliferationin vitroand prolong skin graft survivalin vivo. Experimental Hematology. 2002;30(1):42-48
  5. 5. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126(4):663-676 [Epub Aug 10, 2006]
  6. 6. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131(5):861-872
  7. 7. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007;318(5858):1917-1920 [Epub Nov 20, 2007]
  8. 8. McKinney CE. Using induced pluripotent stem cells derived neurons to model brain diseases. Neural Regeneration Research. 2017;12(7):1062-1067. DOI: 10.4103/1673-5374.211180
  9. 9. Anderson RH, Francis KR. Modeling rare diseases with induced pluripotent stem cell technology. Molecular and Cell Probes. 2018;40:52-59. DOI: 10.1016/j.mcp.2018.01.001
  10. 10. Aasen T, Raya A, Barrero MJ, Garreta E, Consiglio A, Gonzalez F, et al. Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes. Nature Biotechnology. 2008;26(11):1276-1284. DOI: 10.1038/nbt.1503
  11. 11. Ahuja CS, Wilson JR, Nori S, Kotter MRN, Druschel C, Curt A, et al. Traumatic spinal cord injury. Nature Reviews Disease Primers. 2017;3:17018. DOI: 10.1038/nrdp.2017.18
  12. 12. Kwon BK, Liu J, Messerer C, Kobayashi NR, McGraw J, Oschipok L, et al. Survival and regeneration of rubrospinal neurons 1 year after spinal cord injury. Proceedings of the National Academy of Sciences of the United States of America. 2002;99(5):3246-3251 [Epub Feb 26, 2002]
  13. 13. Chen MS, Huber AB, van der Haar ME, et al. Nogo-A is a myelin-associated neurite outgrowth inhibitor and an antigen for monoclonal antibody IN-1. Nature. 2000;403(6768):434-439
  14. 14. Freund P, Schmidlin E, Wannier T, et al. Nogo-A-specific antibody treatment enhances sprouting and functional recovery after cervical lesion in adult primates. Nature Medicine. 2006;12(7):790-792
  15. 15. Cafferty WB, Duffy P, Huebner E, Strittmatter SM. MAG and Omgp synergize with Nogo-A to restrict axonal growth and neurological recovery after spinal cord trauma. The Journal of Neuroscience. 2010;30(20):6825-6837
  16. 16. DeBellard ME, Tang S, Mukhopadhyay G, Shen YJ, Filbin MT. Myelin-associated glycoprotein inhibits axonal regeneration from a variety of neurons via interaction with a sialoglycoprotein. Molecular and Cellular Neurosciences. 1996;7(2):89-101
  17. 17. Barton WA, Liu BP, Tzvetkova D, et al. Structure and axon outgrowth inhibitor binding of the Nogo-66 receptor and related proteins. The EMBO Journal. 2003;22(13):3291-3302
  18. 18. McKeon RJ, Schreiber RC, Rudge JS, Silver J. Reduction of neurite outgrowth in a model of glial scarring following CNS injury is correlated with the expression of inhibitory molecules on reactive astrocytes. The Journal of Neuroscience. 1991;11(11):33983411
  19. 19. Woolfe F, Waxman SG, Hains BC. In silico modeling of axonal reconnection within a discrete fiber tract after spinal cord injury. Journal of Neurotrauma. 2007;24(2):421-432
  20. 20. Dietz V, Curt A. Neurological aspects of spinal-cord repair: Promises and challenges. Lancet Neurology. 2006;5(8):688-694
  21. 21. Friedli L, Rosenzweig ES, Barraud Q, Schubert M, Dominici N, Awai L, et al. Pronounced species divergence in corticospinal tract reorganization and functional recovery after lateralized spinal cord injury favors primates. Science Translational Medicine. 2015;7(302):302ra134. DOI: 10.1126/scitranslmed.aac5811
  22. 22. Assinck P, Duncan GJ, Hilton BJ, Plemel JR, Tetzlaff W. Cell transplantation therapy for spinal cord injury. Nature Neuroscience. 2017;20(5):637-647. DOI: 10.1038/nn.4541
  23. 23. Ahuja CS, Fehlings M. Concise review: Bridging the gap: Novel neuroregenerative and neuroprotective strategies in spinal cord injury. Stem Cells Translational Medicine. 2016;5(7):914-924
  24. 24. Vaquero J, Zurita M, Rico MA, Bonilla C, Aguayo C, Fernández C, et al. Repeated subarachnoid administrations of autologous mesenchymal stromal cells supported in autologous plasma improve quality of life in patients suffering incomplete spinal cord injury. Cytotherapy. 2017;19(3):349-359
  25. 25. Oh SK, Choi KH, Yoo JY, Kim DY, Kim SJ, Jeon SR. A phase III clinical trial showing limited efficacy of autologous mesenchymal stem cell therapy for spinal cord injury. Neurosurgery. 2016;78(3):436-447
  26. 26. Kjell J, Olson L. Rat models of spinal cord injury: From pathology to potential therapies. Disease Models & Mechanisms. 2016;9(10):1125-1137
  27. 27. Takahashi A, Nakajima H, Uchida K, Takeura N, Honjoh K, Watanabe S, et al. Comparison of mesenchymal stromal cells isolated from murine adipose tissue and bone marrow in the treatment of spinal cord injury. Cell Transplantation. 2018;27(7):1126-1139. DOI: 10.1177/0963689718780309
  28. 28. Wang N, Xiao Z, Zhao Y, Wang B, Li X, Li J, et al. Collagen scaffold combined with human umbilical cord-derived mesenchymal stem cells promote functional recovery after scar resection in rats with chronic spinal cord injury. Journal of Tissue Engineering and Regenerative Medicine. 2018;12(2):e1154-e1163
  29. 29. Nicola FDC, Marques MR, Odorcyk F, Arcego DM, Petenuzzo L, Aristimunha D, et al. Neuroprotector effect of stem cells from human exfoliated deciduous teeth transplanted after traumatic spinal cord injury involves inhibition of early neuronal apoptosis. Brain Research. 2017;1663:95-105
  30. 30. Ziegler MD, Hsu D, Takeoka A, Zhong H, Ramón-Cueto A, Phelps PE, et al. Further evidence of olfactory ensheathing glia facilitating axonal regeneration after a complete spinal cord transection. Experimental Neurology. 2011;229(1):109-119
  31. 31. Bunge MB, Monje PV, Khan A, Wood PM. From transplanting Schwann cells in experimental rat spinal cord injury to their transplantation into human injured spinal cord in clinical trials. Progress in Brain Research. 2017;231:107-133
  32. 32. Rosenzweig ES, Brock JH, Lu P, Kumamaru H, Salegio EA, Kadoya K, et al. Restorative effects of human neural stem cell grafts on the primate spinal cord. Nature Medicine. 2018;24(4):484-490
  33. 33. Vawda R, Fehlings MG. Mesenchymal cells in the treatment of spinal cord injury: Current & future perspectives. Current Stem Cell Research & Therapy. 2013;8(1):25-38
  34. 34. Matyas JJ, Stewart AN, Goldsmith A, Nan Z, Skeel RL, Rossignol J, et al. Effects of bone-marrow-derived MSC transplantation on functional recovery in a rat model of spinal cord injury: Comparisons of transplant locations and cell concentrations. Cell Transplantation. 2017;26(8):1472-1482
  35. 35. Torres-Espín A, Redondo-Castro E, Hernandez J, Navarro X. Immunosuppression of allogenic mesenchymal stem cells transplantation after spinal cord injury improves graft survival and beneficial outcomes. Journal of Neurotrauma. 2015;32(6):367-380. DOI: 10.1089/neu.2014.356236
  36. 36. Zhu Y, Uezono N, Yasui T, Nakashima K. Neural stem cell therapy aiming at better functional recovery after spinal cord injury. Developmental Dynamics. 2018;247(1):75-84. DOI: 10.1002/dvdy.24558. 37
  37. 37. Meletis K, Barnabé-Heider F, Carlén M, Evergren E, Tomilin N, Shupliakov O, et al. Spinal cord injury reveals multilineage differentiation of ependymal cells. PLoS Biology. 2008;6:e182
  38. 38. Moreno-Manzano V, Rodríguez-Jiménez FJ, García-Roselló M, Laínez S, Erceg S, Calvo MT, et al. Activated spinal cord ependymal stem cells rescue neurological function. Stem Cells. 2009;27:733-743
  39. 39. Requejo-Aguilar R, Alastrue-Agudo A, Cases-Villar M, Lopez-Mocholi E, England R, Vicent MJ, et al. Combined polymer-curcumin conjugate and ependymal progenitor/stem cell treatment enhances spinal cord injury functional recovery. Biomaterials. 2017;113:18-30
  40. 40. Gómez-Villafuertes R, Rodríguez-Jiménez FJ, Alastrue-Agudo A, Stojkovic M, Miras-Portugal MT, Moreno-Manzano V. Purinergic receptors in spinal cord-derived ependymal stem/progenitor cells and their potential role in cell-based therapy for spinal cord injury. Cell Transplantation. 2015;24:1493-1509
  41. 41. Curtis E, Martin JR, Gabel B, Sidhu N, Rzesiewicz TK, Mandeville R, Van Gorp S, et al. A first-in-human, phase I study of neural stem cell transplantation for chronic spinal cord injury. Cell Stem Cell. 2018;22(6):941-950.e6
  42. 42. Shi Y, Su J, Roberts AI, Shou P, Rabson AB, Ren G. How mesenchymal stem cells interact with tissue immune responses. Trends in Immunology. 2012;33(3):136-143. DOI: 10.1016/j.it.2011.11.004
  43. 43. Uccelli A, de Rosbo NK. The immunomodulatory function of mesenchymal stem cells: Mode of action and pathways. Annals of the New York Academy of Sciences. 2015;1351:114-126. DOI: 10.1111/nyas.12815
  44. 44. Wang M, Yuan Q, Xie L. Mesenchymal stem cell-based immunomodulation: Properties and clinical application. Stem Cells International. 2018;2018:3057624. DOI: 10.1155/2018/3057624
  45. 45. Wang LT, Ting CH, Yen ML, Liu KJ, Sytwu HK, Wu KK, et al. Human mesenchymal stem cells (MSCs) for treatment towards immune- and inflammation-mediated diseases: Review of current clinical trials. Journal of Biomedical Science. 2016;23(1):76
  46. 46. Xiao J, Yang R, Biswas S, Qin X, Zhang M, Deng W. Mesenchymal stem cells and induced pluripotent stem cells as therapies for multiple sclerosis. International Journal of Molecular Sciences. 2015;16(5):9283-9302. DOI: 10.3390/ijms16059283
  47. 47. Freedman MS, Bar-Or A, Atkins HL, Karussis D, Frassoni F, Lazarus H, et al. The therapeutic potential of mesenchymal stem cell transplantation as a treatment for multiple sclerosis: Consensus report of the International MSCT Study Group. Multiple Sclerosis. 2010;16(4):503-510. DOI: 10.1177/1352458509359727. 20086020
  48. 48. Scolding NJ, Pasquini M, Reingold SC, Cohen JA. International Conference on Cell-Based Therapies for Multiple Sclerosis. Cell-based therapeutic strategies for multiple sclerosis. Brain. 2017;140(11):2776-2796. DOI: 10.1093/brain/awx154
  49. 49. Laterza C, Merlini A, De Feo D, Ruffini F, Menon R, Onorati M, et al. iPSC-derived neural precursors exert a neuroprotective role in immune-mediated demyelination via the secretion of LIF. Nature Communications. 2013;4:2597. DOI: 10.1038/ncomms3597
  50. 50. Marriott MP, Emery B, Cate HS, Binder MD, Kemper D, Wu Q, et al. Leukemia inhibitory factor signaling modulates both central nervous system demyelination and myelin repair. Glia. 2008;56(6):686-698. DOI: 10.1002/glia.20646
  51. 51. Butzkueven H, Emery B, Cipriani T, Marriott MP, Kilpatrick TJ. Endogenous leukemia inhibitory factor production limits autoimmune demyelination and oligodendrocyte loss. Glia. 2006;53(7):696-703
  52. 52. Carruthers BM, van de Sande MI, De Meirleir KL, Klimas NG, Broderick G, Mitchell T, et al. Myalgic encephalomyelitis: International consensus criteria. Journal of Internal Medicine. 2011;270(4):327-338. DOI: 10.1111/j.1365-2796.2011.02428.x [Epub Aug 22, 2011]. (Review. Erratum in: Journal of Internal Medicine. 2017;282(4):353)
  53. 53. Hornig M, Gottschalk CG, Eddy ML, Che X, Ukaigwe JE, Peterson DL, et al. Immune network analysis of cerebrospinal fluid in myalgic encephalomyelitis/chronic fatigue syndrome with atypical and classical presentations. Translational Psychiatry. 2017;7(4):e1080
  54. 54. Broderick G, Fuite J, Kreitz A, Vernon SD, Klimas N, Fletcher MA. A formal analysis of cytokine networks in chronic fatigue syndrome. Brain, Behavior, and Immunity. 2010;24(7):1209-1217. DOI: 10.1016/j.bbi.2010.04.012
  55. 55. Brenu EW, Hardcastle SL, Atkinson GM, van Driel ML, Kreijkamp-Kaspers S, Ashton KJ, et al. Natural killer cells in patients with severe chronic fatigue syndrome. Autoimmunity Highlights. 2013;4(3):69-80
  56. 56. Institute of Medicine, Committee on the Diagnostic Criteria for Myalgic Encephalomyelitis/Chronic Fatigue Syndrome, Board on the Health of Select Populations. Beyond Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: Redefining an Illness. Washington (DC): National Academies Press; 2015. 304p
  57. 57. Clayton EW. Beyond myalgic encephalomyelitis/chronic fatigue syndrome: An IOM report on redefining an illness. Journal of the American Medical Association. 2015;313(11):1101-1102
  58. 58. Capelli E, Zola R, Lorusso L, Venturini L, Sardi F, Ricevuti G. Chronic fatigue syndrome/myalgic encephalomyelitis: An update. International Journal of Immunopathology and Pharmacology. 2010;23(4):981-989
  59. 59. Sotzny F, Blanco J, Capelli E, Castro-Marrero J, Steiner S, Murovska M, et al. Myalgic encephalomyelitis/chronic fatigue syndrome—Evidence for an autoimmune disease. Autoimmunity Reviews. 2018;17(6):601-609. DOI: 10.1016/j.autrev.2018.01.009
  60. 60. Gow JW, Simpson K, Behan PO, Chaudhuri A, McKay IC, Behan WM. Antiviral pathway activation in patients with chronic fatigue syndrome and acute infection. Clinical Infectious Diseases. 2001;33(12):2080-2081 [Epub Nov 6, 2001]
  61. 61. Roerink ME, Roerink SHPP, Skoluda N, van der Schaaf ME, Hermus ARMM, van der Meer JWM, et al. Hair and salivary cortisol in a cohort of women with chronic fatigue syndrome. Hormones and Behavior. 2018;103:1-6. DOI: 10.1016/j.yhbeh.2018.05.016
  62. 62. Sedghamiz H, Morris M, Craddock TJA, Whitley D, Broderick G. High-fidelity discrete modeling of the HPA axis: A study of regulatory plasticity in biology. BMC Systems Biology. 2018;12(1):76. DOI: 10.1186/s12918-018-0599-1
  63. 63. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8(4):315-317
  64. 64. Bourin P, Bunnell BA, Casteilla L, Dominici M, Katz AJ, March KL, et al. Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells: A joint statement of the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT). Cytotherapy. 2013;15(6):641-648. DOI: 10.1016/j.jcyt.2013.02.006
  65. 65. Hass R, Kasper C, Böhm S, Jacobs R. Different populations and sources of human mesenchymal stem cells (MSC): A comparison of adult and neonatal tissue-derived MSC. Cell Communication and Signaling: CCS. 2011;9:12. DOI: 10.1186/1478-811X-9-12
  66. 66. Hoogduijn MJ, de Witte SF, Luk F, van den Hout-van Vroonhoven MC, Ignatowicz L, Catar R, et al. Effects of freeze-thawing and intravenous infusion on mesenchymal stromal cell gene expression. Stem Cells and Development. 2016;25(8):586-597. DOI: 10.1089/scd.2015.0329
  67. 67. Jossen V, van den Bos C, Eibl R, Eibl D. Manufacturing human mesenchymal stem cells at clinical scale: Process and regulatory challenges. Applied Microbiology and Biotechnology. 2018;102(9):3981-3994. DOI: 10.1007/s00253-018-8912-x
  68. 68. de Soure AM, Fernandes-Platzgummer A, da Silva CL, Cabral JM. Scalable microcarrier-based manufacturing of mesenchymal stem/stromal cells. Journal of Biotechnology. 2016;236:88-109. DOI: 10.1016/j.jbiotec.2016.08.007
  69. 69. Giancola R, Bonfini T, Iacone A. Cell therapy: cGMP facilities and manufacturing. Muscle, Ligaments and Tendons Journal 2012;2(3):243-247 [Print Jul 2012]
  70. 70. Soleimani M, Nadri S. A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow. Nature Protocols. 2009;4(1):102-106. DOI: 10.1038/nprot.2008.221
  71. 71. Zuk PA, Zhu M, Ashjian P, De Ugarte DA, Huang JI, Mizuno H, et al. Human adipose tissue is a source of multipotent stem cells. Molecular Biology of the Cell. 2002;13(12):4279-4295
  72. 72. Nordberg RC, Loboa EG. Our fat future: Translating adipose stem cell therapy. Stem Cells Translational Medicine. 2015;4(9):974-979. DOI: 10.5966/sctm.2015-0071
  73. 73. Van den Bos C. Off the beaten track—regulatory changes. European Biopharmaceutical Reviews. 2012;165(winter):32-36
  74. 74. Halme DG, Kessler DA. FDA regulation of stem-cell-based therapies. New England Journal of Medicine. 2006;355:409-415. DOI: 10.1056/NEJMhpr063086
  75. 75. Yi T, Kim SN, Lee HJ, Kim J, Cho YK, Shin DH, et al. Manufacture of clinical-grade human clonal mesenchymal stem cell products from single colony forming unit-derived colonies based on the subfractionation culturing method. Tissue Engineering. Part C, Methods. 2015;21(12):1251-1262. DOI: 10.1089/ten.TEC.2015.0017
  76. 76. Dessels C, Potgieter M, Pepper MS. Making the switch: Alternatives to fetal bovine serum for adipose-derived stromal cell expansion. Frontiers in Cell and Development Biology. 2016;4:115
  77. 77. Horwitz EM, Gordon PL, Koo WKK, Marx JC, Neel MD, McNall RY, et al. Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: Implications for cell therapy of bone. Proceedings of the National Academy of Sciences of the United States of America. 2002;99:8932-8937. DOI: 10.1073/pnas.132252399
  78. 78. Selvaggi TA, Walker RE, Fleisher TA. Development of antibodies to fetal calf serum with arthus-like reactions in human immunodeficiency virus-infected patients given syngeneic lymphocyte infusions. Blood. 1997;89:776-779
  79. 79. Mackensen A, Dräger R, Schlesier M, Mertelsmann R, Lindemann A. Presence of IgE antibodies to bovine serum albumin in a patient developing anaphylaxis after vaccination with human peptide-pulsed dendritic cells. Cancer Immunology, Immunotherapy. 2000;49:152-156
  80. 80. Josh F, Kobe K, Tobita M, Tanaka R, Suzuki K, Ono K, et al. Accelerated and safe proliferation of human adipose-derived stem cells in medium supplemented with human serum. Journal of Nippon Medical School. 2012;79:444-452. DOI: 10.1272/jnms.79.444
  81. 81. Koellensperger E, Bollinger N, Dexheimer V, Gramley F, Germann G, Leimer U. Choosing the right type of serum for different applications of human adipose tissue-derived stem cells: Influence on proliferation and differentiation abilities. Cytotherapy. 2014;16(6):789-799. DOI: 10.1016/j.jcyt.2014.01.007
  82. 82. Cho H, Lee A, Kim K. The effect of serum types on Chondrogenic differentiation of adipose-derived stem cells. Biomater Research. 2018;22:6. DOI: 10.1186/s40824-018-0116-z
  83. 83. Kocaoemer A, Kern S, Klüter H, Bieback K. Human AB serum and thrombin-activated platelet-rich plasma are suitable alternatives to fetal calf serum for the expansion of mesenchymal stem cells from adipose tissue. Stem Cells. 2007;25(5):1270-1278
  84. 84. Pierce J, Benedetti E, Preslar A, Jacobson P, Jin P, Stroncek DF, et al. Comparative analyses of industrial-scale human platelet lysate preparations. Transfusion. 2017;57(12):2858-2869. DOI: 10.1111/trf.14324
  85. 85. Saury C, Lardenois A, Schleder C, Leroux I, Lieubeau B, David L, et al. Human serum and platelet lysate are appropriate xeno-free alternatives for clinical-grade production of human MuStem cell batches. Stem Cell Research & Therapy. 2018;9(1):128. DOI: 10.1186/s13287-018-0852-y
  86. 86. Bieback K, Hecker A, Kocaömer A, Lannert H, Schallmoser K, Strunk D, et al. Human alternatives to fetal bovine serum for the expansion of mesenchymal stromal cells from bone marrow. Stem Cells. 2009;27(9):2331-2341. DOI: 10.1002/stem.139
  87. 87. Tang H, Xiang Y, Jiang X, Ke Y, Xiao Z, Guo Y, et al. Dual expression of hTERT and VEGF prolongs life span and enhances angiogenic ability of aged BMSCs. Biochemical and Biophysical Research Communications. 2013;440:502-508. DOI: 10.1016/j.bbrc.2013.09.053
  88. 88. Turinetto V, Vitale E, Giachino C. Senescence in human mesenchymal stem cells: functional changes and implications in stem cell-based therapy. International Journal of Molecular Sciences. 2016;17(7). pii: E1164. DOI: 10.3390/ijms17071164
  89. 89. Peng Y, Huang S, Wu Y, Cheng B, Nie X, Liu H, et al. Platelet rich plasma clot releasate preconditioning induced PI3K/AKT/NFκB signaling enhances survival and regenerative function of rat bone marrow mesenchymal stem cells in hostile microenvironments. Stem Cells and Development. 2013;22(24):3236-3251. DOI: 10.1089/scd.2013.0064
  90. 90. Mellado-López M, Griffeth RJ, Meseguer-Ripolles J, Cugat R, García M, Moreno-Manzano V. Plasma rich in growth factors induces cell proliferation, migration, differentiation, and cell survival of adipose-derived stem cells. Stem Cells International. 2017;2017:5946527. DOI: 10.1155/2017/5946527
  91. 91. Usta SN, Scharer CD, Xu J, Frey TK, Nash RJ. Chemically defined serum-free and xeno-free media for multiple cell lineages. Annals of Translational Medicine. 2014;2:97. DOI: 10.3978/j.issn.2305-5839.2014.09.05
  92. 92. Patrikoski M, Juntunen M, Boucher S, Campbell A, Vemuri MC, Mannerström B, et al. Development of fully defined xeno-free culture system for the preparation and propagation of cell therapy compliant human adipose stem cells. Stem Cell Research & Therapy. 2013;4:27. DOI: 10.1186/scrt175
  93. 93. Oikonomopoulos A, vanDeen WK, Manansala AR, Lacey PN, Tomakili TA, Ziman A, et al. Optimization of human mesenchymal stem cell manufacturing: The effects of animal/xeno-free media. Scientific Reports. 2015;5:16570. DOI: 10.1038/srep16570
  94. 94. Lindroos B, Boucher S, Chase L, Kuokkanen H, Huhtala H, Haataja R, et al. Serum-free, xeno-free culture media maintain the proliferation rate and multipotentiality of adipose stem cellsin vitro. Cytotherapy. 2009;11(7):958-972. DOI: 10.3109/14653240903233081
  95. 95. Chen C, Tang Q, Zhang Y, Yu M, Jing W, Tian W. Physioxia: A more effective approach for culturing human adipose-derived stem cells for cell transplantation. Stem Cell Research & Therapy. 2018;9(1):148. DOI: 10.1186/s13287-018-0891-4
  96. 96. Okolicsanyi RK, Camilleri ET, Oikari LE, Yu C, Cool SM, van Wijnen AJ, et al. Human mesenchymal stem cells retain multilineage differentiation capacity including neural marker expression after extendedIn vivoexpansion. PLoS One. 2015;10(9):e0137255. DOI: 10.1371/journal.pone.0137255 (eCollection 2015)
  97. 97. Zachar L, Bačenková D, Rosocha J. Activation, homing, and role of the mesenchymal stem cells in the inflammatory environment. Journal of Inflammation Research. 2016;9:231-240. DOI: 10.2147/JIR.S121994 (eCollection 2016)
  98. 98. Ma OK, Chan KH. Immunomodulation by mesenchymal stem cells: Interplay between mesenchymal stem cells and regulatory lymphocytes. World Journal of Stem Cells. 2016;8(9):268-278. DOI: 10.4252/wjsc.v8.i9.268
  99. 99. Li W, Ren G, Huang Y, Su J, Han Y, Li J, et al. Mesenchymal stem cells: A double-edged sword in regulating immune responses. Cell Death and Differentiation. 2012;19(9):1505-1513. DOI: 10.1038/cdd.2012.26
  100. 100. Prockop DJ, Oh JY. Mesenchymal stem/stromal cells (MSCs): Role as guardians of inflammation. Molecular Therapy. 2012;20(1):14-20. DOI: 10.1038/mt.2011.211
  101. 101. Le Blanc K, Davies LC. Mesenchymal stromal cells and the innate immune response. Immunology Letters. 2015;168(2):140-146. DOI: 10.1016/j.imlet.2015.05.004
  102. 102. Hoogduijn MJ, Roemeling-van Rhijn M, Engela AU, Korevaar SS, Mensah FK, Franquesa M, et al. Mesenchymal stem cells induce an inflammatory response after intravenous infusion. Stem Cells and Development. 2013;22(21):2825-2835. DOI: 10.1089/scd.2013.0193
  103. 103. Ulivi V, Tasso R, Cancedda R, Descalzi F. Mesenchymal stem cell paracrine activity is modulated by platelet lysate: Induction of an inflammatory response and secretion of factors maintaining macrophages in a proinflammatory phenotype. Stem Cells and Development. 2014;23(16):1858-1869. DOI: 10.1089/scd.2013.0567
  104. 104. de Witte SF, Franquesa M, Baan CC, Hoogduijn MJ. Toward development of mesenchymal stem cells for immunomodulatory therapy. Frontiers in Immunology. 2016;6:648. DOI: 10.3389/fimmu.2015.00648
  105. 105. Moll G, Jitschin R, von Bahr L, Rasmusson-Duprez I, Sundberg B, Lönnies L, et al. Mesenchymal stromal cells engage complement and complement receptor bearing innate effector cells to modulate immune responses. PLoS One. 2011;6(7):e21703. DOI: 10.1371/journal.pone.0021703
  106. 106. Tu Z, Li Q, Bu H, Lin F. Mesenchymal stem cells inhibit complement activation by secreting factor H. Stem Cells and Development. 2010;19(11):1803-1809. DOI: 10.1089/scd.2009.0418
  107. 107. Kadoya K, Lu P, Nguyen K, Lee-Kubli C, Kumamaru H, Yao L, et al. Spinal cord reconstitution with homologous neural grafts enables robust corticospinal regeneration. Nature Medicine. 2016;22(5):479-487
  108. 108. Mothe AJ, Tator CH. Advances in stem cell therapy for spinal cord injury. The Journal of Clinical Investigation. 2012;122(11):3824-3834
  109. 109. Matsui T, Akamatsu W, Nakamura M, Okano H. Regeneration of the damaged central nervous system through reprogramming technology: Basic concepts and potential application for cell replacement therapy. Experimental Neurology. 2014;260:12-18
  110. 110. Mothe AJ, Zahir T, Santaguida C, Cook D, Tator CH. Neural stem/progenitor cells from the adult human spinal cord are multipotent and self-renewing and differentiate after transplantation. PLoS One. 2011;6(11):e27079. DOI: 10.1371/journal.pone.0027079
  111. 111. Lukovic D, Diez Lloret A, Stojkovic P, Rodríguez-Martínez D, Perez Arago MA, Rodriguez-Jimenez FJ, et al. Highly efficient neural conversion of human pluripotent stem cells in adherent and animal-free conditions. Stem Cells Translational Medicine. 2017;6(4):1217-1226. DOI: 10.1002/sctm.16-0371
  112. 112. Tsuji O, Miura K, Okada Y, Fujiyoshi K, Mukaino M, Nagoshi N, et al. Therapeutic potential of appropriately evaluated safeinduced pluripotent stem cells for spinal cord injury. Proceedings of the National Academy of Sciences of the United States of America. 2010;107(28):12704-12709
  113. 113. Nori S, Okada Y, Yasuda A, Tsuji O, Takahashi Y, Kobayashi Y, et al. Grafted human-induced pluripotent stem cell derived neurospheres promote motor functional recovery after spinal cord injury in mice. Proceedings of the National Academy of Sciences of the United States of America. 2011;108(40):16825-16830
  114. 114. Fujimoto Y, Abematsu M, Falk A, Tsujimura K, Sanosaka T, Juliandi B, et al. Treatment of mouse model of spinal cord injury by transplantation of human induced pluripotent stem cell-derived long-term self-renewing neuroepithelial-like stem cells. Stem Cells. 2012;30(6):1163-1173
  115. 115. López-Serrano C, Torres-Espín A, Hernández J, Alvarez-Palomo AB, Requena J, Gasull X, et al. Effects of the post-spinal cord injury microenvironment on the differentiation capacity of human neural stem cells derived from induced pluripotent stem cells. Cell Transplantation. 2016;25(10):1833-1852
  116. 116. Säljö K, Barone A, Mölne J, Rydberg L, Teneberg S, Breimer ME. HLA and Histo-blood group antigen expression in human pluripotent stem cells and their derivatives. Scientific Reports. 2017;7(1):13072
  117. 117. Badja C, Maleeva G, El-Yazidi C, Barruet E, Lasserre M, Tropel P, et al. Efficient and cost-effective generation of mature neurons from human induced pluripotent stem cells. Stem Cells Translational Medicine. 2014;3(12):1467-1472. DOI: 10.5966/sctm.2014-0024
  118. 118. Qu X, Liu T, Song K, Li X, Ge D. Induced pluripotent stem cells generated from human adipose-derived stem cells using a non-viral polycistronic plasmid in feeder-free conditions. PLoS One. 2012;7(10):e48161. DOI: 10.1371/journal.pone.0048161
  119. 119. Eminli S, Foudi A, Stadtfeld M, Maherali N, Ahfeldt T, Mostoslavsky G, et al. Differentiation stage determines potential of hematopoietic cells for reprogramming into induced pluripotent stem cells. Nature Genetics. 2009;41(9):968-976. DOI: 10.1038/ng.428
  120. 120. Kim JB, Greber B, Araúzo-Bravo MJ, Meyer J, Park KI, Zaehres H, et al. Direct reprogramming of human neural stem cells by OCT4. Nature. 2009;461(7264):649-643. DOI: 10.1038/nature08436
  121. 121. Anokye-Danso F, Trivedi CM, Juhr D, Gupta M, Cui Z, Tian Y, et al. Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency. Cell Stem Cell. 2011;8(4):376-388. DOI: 10.1016/j.stem.2011.03.001 (Erratum in: Cell Stem Cell. 2012;11(6):853)
  122. 122. Khazaei M, Ahuja CS, Fehlings MG. Induced pluripotent stem cells for traumatic spinal cord injury. Frontiers in Cell and Development Biology. 2017;4:152. DOI: 10.3389/fcell.2016.00152
  123. 123. Ban H, Nishishita N, Fusaki N, Tabata T, Saeki K, Shikamura M, et al. Efficient generation of transgene-free human induced pluripotent stem cells (iPSCs) by temperature-sensitive Sendai virus vectors. Proceedings of the National Academy of Sciences of the United States of America. 2011;108(34):14234-14239. DOI: 10.1073/pnas.1103509108
  124. 124. Zhou W, Freed CR. Adenoviral gene delivery can reprogram human fibroblasts to induced pluripotent stem cells. Stem Cells. 2009;27(11):2667-2674. DOI: 10.1002/stem.201
  125. 125. Maherali N, Hochedlinger K. Guidelines and techniques for the generation of induced pluripotent stem cells. Cell Stem Cell. 2008;3(6):595-605. DOI: 10.1016/j.stem.2008.11.008
  126. 126. González F, Boué S, Izpisúa Belmonte JC. Methods for making induced pluripotent stem cells: Reprogramming à la carte. Nature Reviews. Genetics. 2011;12(4):231-242. DOI: 10.1038/nrg2937
  127. 127. Brouwer M, Zhou H, Nadif KN. Choices for induction of pluripotency: Recent developments in human induced pluripotent stem cell reprogramming strategies. Stem Cell Reviews. 2016;12(1):54-72. DOI: 10.1007/s12015-015-9622-8
  128. 128. Fandel TM, Trivedi A, Nicholas CR, Zhang H, Chen J, Martinez AF, et al. Transplanted human stem cell-derived interneuron precursors mitigate mouse bladder dysfunction and central neuropathic pain after spinal cord injury. Cell Stem Cell. 2016;19(4):544-557. DOI: 10.1016/j.stem.2016.08.020
  129. 129. Orlova VV, Drabsch Y, Freund C, Petrus-Reurer S, van den Hil FE, Muenthaisong S, et al. Functionality of endothelial cells and pericytes from human pluripotent stem cells demonstrated in cultured vascular plexus and zebrafish xenografts. Arteriosclerosis, Thrombosis, and Vascular Biology. 2014;34(1):177-186. DOI: 10.1161/ATVBAHA.113.302598
  130. 130. Yu C, Griffiths LR, Haupt LM. Exploiting Heparan Sulfate proteoglycans in human neurogenesis-controlling lineage specification and fate. Frontiers in Integrative Neuroscience. 2017;11:28. DOI: 10.3389/fnint.2017.00028
  131. 131. Rodríguez-Jimenez FJ, Alastrue-Agudo A, Erceg S, Stojkovic M, Moreno-Manzano V. FM19G11 favors spinal cord injury regeneration and stem cell self-renewal by mitochondrial uncoupling and glucose metabolism induction. Stem Cells. 2012;30(10):2221-2233
  132. 132. Alastrue-Agudo A, Rodriguez-Jimenez FJ, Mocholi EL, De Giorgio F, Erceg S, Moreno-Manzano V. FM19G11 and ependymal progenitor/stem cell combinatory treatment enhances neuronal preservation and oligodendrogenesis after severe spinal cord injury. International Journal of Molecular Sciences. 2018;19(1). pii: E200. DOI: 10.3390/ijms19010200
  133. 133. Carrero R, Cerrada I, Lledó E, Dopazo J, García-García F, Rubio MP, et al. IL1β induces mesenchymal stem cells migration and leucocyte chemotaxis through NF-κB. Stem Cell Reviews. 2012;8(3):905-916. DOI: 10.1007/s12015-012-9364-9
  134. 134. Hu C, Li L. Preconditioning influences mesenchymal stem cell propertiesin vitroandin vivo. Journal of Cellular and Molecular Medicine. 2018;22(3):1428-1442. DOI: 10.1111/jcmm.13492
  135. 135. Wang J, Li Z, Zhang Y, Liu X, Chen L, Chen Y. CX43 change in LPS preconditioning against apoptosis of mesenchymal stem cells induced by hypoxia and serum deprivation is associated with ERK signaling pathway. Molecular and Cellular Biochemistry. 2013;380(1-2):267-275. DOI: 10.1007/s11010-013-1683-x
  136. 136. Liao W, Pham V, Liu L, et al. Mesenchymal stem cells engineered to express selectin ligands and IL-10 exert enhanced therapeutic efficacy in murine experimental autoimmune encephalomyelitis. Biomaterials. 2016;77:87-97. DOI: 10.1016/j.biomaterials.2015.11.005
  137. 137. Berglund AK, Fortier LA, Antczak DF, Schnabel LV. Immunoprivileged no more: Measuring the immunogenicity of allogeneic adult mesenchymal stem cells. Stem Cell Research & Therapy. 2017;8(1):288. DOI: 10.1186/s13287-017-0742-8
  138. 138. Lohan P, Treacy O, Griffin MD, Ritter T, Ryan AE. Anti-donor immune responses elicited by allogeneic mesenchymal stem cells and their extracellular vesicles: Are we still learning? Frontiers in Immunology. 2017;8:1626. DOI: 10.3389/fimmu.2017.01626
  139. 139. Hofer HR, Tuan RS. Secreted trophic factors of mesenchymal stem cells support neurovascular and musculoskeletal therapies. Stem Cell Research & Therapy. 2016;7:131. DOI: 10.1186/s13287-016-0394-0
  140. 140. Cizkova D, Cubinkova V, Smolek T, Murgoci AN, Danko J, Vdoviakova K, et al. Localized intrathecal delivery of mesenchymal stromal cells conditioned medium improves functional recovery in a rat model of spinal cord injury. International Journal of Molecular Sciences. 2018;19(3). pii: E870. DOI: 10.3390/ijms19030870 (Erratum in: International Journal of Molecular Sciences. 2018;19(7))
  141. 141. Haynesworth SE, Baber MA, Caplan AI. Cytokine expression by human marrow-derived mesenchymal progenitor cellsin vitro: Effects of dexamethasone and IL-1 alpha. Journal of Cellular Physiology. 1996;166(3):585-592
  142. 142. Keshtkar S, Azarpira N, Ghahremani MH. Mesenchymal stem cell-derived extracellular vesicles: Novel frontiers in regenerative medicine. Stem Cell Research & Therapy. 2018;9(1):63. DOI: 10.1186/s13287-018-0791-7
  143. 143. Lötvall J, Hill AF, Hochberg F, Buzás EI, Di Vizio D, Gardiner C, et al. Minimal experimental requirements for definition of extracellular vesicles and their functions: A position statement from the International Society for Extracellular Vesicles. Journal of Extracellular Vesicles. 2014;3:26913. DOI: 10.3402/jev.v3.26913
  144. 144. Kordelas L, Rebmann V, Ludwig AK, Radtke S, Ruesing J, Doeppner TR, et al. MSC-derived exosomes: A novel tool to treat therapy-refractory graft-versus-host disease. Leukemia. 2014;28:970-973. DOI: 10.1038/leu.2014.41
  145. 145. Drommelschmidt K, Serdar M, Bendix I, Herz J, Bertling F, Prager S, et al. Mesenchymal stem cell-derived extracellular vesicles ameliorate inflammation-induced preterm brain injury. Brain, Behavior, and Immunity. 2017;60:220-232. DOI: 10.1016/j.bbi.2016.11.011
  146. 146. Ruppert KA, Nguyen TT, Prabhakara KS, Toledano Furman NE, Srivastava AK, et al. Human mesenchymal stromal cell-derived extracellular vesicles modify microglial response and improve clinical outcomes in experimental spinal cord injury. Scientific Reports. 2018;8(1):480. DOI: 10.1038/s41598-017-18867-w
  147. 147. Lener T, Gimona M, Aigner L, Borger V, Buzas E, Camussi G, et al. Applying extracellular vesicles based therapeutics in clinical trials—An ISEV position paper. Journal of Extracellular Vesicles. 2015;4:30087
  148. 148. Gimona M, Pachler K, Laner-Plamberger S, Schallmoser K, Rohde E. Manufacturing of human extracellular vesicle-based therapeutics for clinical use. International Journal of Molecular Sciences. 2017;18(6). pii: E1190. DOI: 10.3390/ijms18061190
  149. 149. Mora EM, Álvarez-Cubela S, Oltra E. Biobanking of exosomes in the era of precision medicine: Are we there yet? International Journal of Molecular Sciences. 2015;17(1). pii: E13. DOI: 10.3390/ijms17010013
  150. 150. García-Contreras M, Vera-Donoso CD, Hernández-Andreu JM, García-Verdugo JM, Oltra E. Therapeutic potential of human adipose-derived stem cells (ADSCs) from cancer patients: A pilot study. PLoS One. 2014;9(11):e113288. DOI: 10.1371/journal.pone.0113288

Written By

Victoria Moreno-Manzano and Elisa Oltra García

Submitted: April 20th, 2018 Reviewed: August 5th, 2018 Published: November 5th, 2018