On the Viability and Potential Value of Stem Cells for Repair and Treatment of Central Neurotrauma: Overview and Speculations

Central neurotrauma, such as spinal cord injury or traumatic brain injury, can damage critical axonal pathways and neurons and lead to partial to complete loss of neural function that is difficult to address in the mature central nervous system. Improvement and innovation in the development, manufacture, and delivery of stem-cell based therapies, as well as the continued exploration of newer forms of stem cells, have allowed the professional and public spheres to resolve technical and ethical questions that previously hindered stem cell research for central nervous system injury. Recent in vitro and in vivo models have demonstrated the potential that reprogrammed autologous stem cells, in particular, have to restore functionality and induce regeneration—while potentially mitigating technical issues of immunogenicity, rejection, and ethical issues of embryonic derivation. These newer stem-cell based approaches are not, however, without concerns and problems of safety, efficacy, use and distribution. This review is an assessment of the current state of the science, the potential solutions that have been and are currently being explored, and the problems and questions that arise from what appears to be a promising way forward (i.e., autologous stem cell-based therapies)—for the purpose of advancing the research for much-needed therapeutic interventions for central neurotrauma.


NEUROTRAUMA: AN OVERVIEW
Neurotrauma is defined as neurological insult that results in the disturbance of neural circuitry through disruption of axonal pathways and/or neural cell damage or loss (1). Injuries to the central nervous system (CNS) include, but are not limited to, spinal cord injury (SCI), traumatic brain injury (TBI), and stroke (1)(2)(3)(4).
The epidemiological impact of neurotrauma is significant. Annually, there are approximately 12,000 cases of SCI in the United States alone (5), with injury being incurred by compression, contusion, laceration and/or partial or complete severing of the cord (6). There are 1.5-2 million cases of traumatic brain injury (TBI) per year in the US, with 75,000-100,000 of these cases being classified as severe (7,8). Approximately 795,000 strokes occur annually in the US, with 87% of these being ischemic and 10% due to intracranial hemorrhage (9). Neurotrauma can incur both short and longterm, partial or complete loss of neurological function, depending on the type and severity of injuries (7,8,10). Pathophysiological features can include sensory and/or autonomic impairment, muscle weakness, and/or decreased control of movement, decreased endurance, muscle spasms, and hypertonicity, and are typically reflective of the site(s) and extent of neurological damage (6)(7)(8)11). Onset of signs and symptoms resulting from neurotrauma can begin immediately following injury, or in some cases (of mild to moderate insult, with iterative neurological involvement) can be latent, and can persist for months, years and/or durably (i.e., -permanently).
The short-and long-term effects of neurotrauma can extend beyond the biophysiological to detrimentally affect quality of life (i.e., employment opportunities, relationships, ability to partake in recreational activities, etc.) (12)(13)(14). Post-trauma care and management of symptoms can also be economically burdensome for individuals and families affected by SCI, TBI or stroke. For example, in the US, the first year of care following an SCI injury, can incur individual costs ranging from US$123,000 to US$423,000 (15). Lifetime costs for an individual injured at 25 years of age have been estimated to reach US$2.7 million, depending on the severity of the injury incurred (16).
While we recognize that SCI, TBI, and stroke each and all have differing therapeutic targets, and methods, and have been, and may be therapeutically approached in distinct ways, herein we take a heterogeneous approach to include all of these conditions in this review, as stem cells are and may be used in a variety of therapeutic applications. As well, we place specific emphasis on SCI, recognizing the particular potential for application of stem cell-based therapies in this context, and acknowledge and address that TBI and stroke have been shown to share certain pathophysiological processes and clinical targets, which have been supportive of stem-cell based interventions (3,17,18).

THE THERAPEUTIC/RECUPERATIVE PROBLEM
Patterns of recovery of function following neurotrauma generally reflect neurons' incapacity for replication (i.e., post-mitotic stability). It is noteworthy that some areas of the brain have shown regenerative capacity (i.e., neurogenesis) (19)(20)(21) through initiation and migration of neural progenitor cells. Regions that have exhibited progenitor cell activation and resulting neurogenesis include the hippocampal subgranular zone (22)(23)(24) and dentate gyrus [(25); however, for contrasting report, see (26)], the periventricular area, the olfactory bulb (27)(28)(29)(30), the subventricular zone (31)(32)(33), and the central canal of the spinal cord (34)(35)(36)(37). However, progenitor cells appear to be isolated to these regions, and are not ubiquitous in the CNS. The majority of mature neurons of the CNS are terminally differentiated (TD), can no longer undergo mitosis, and are considered to be outside of the cell cycle (38). While there is some debate whether neurogenesis may persist into and throughout adulthood in certain brain areas [e.g., the dentate gyrus; (25,26,39)]; such mitotic capacity, even if limited to key periods in development, is certainly not neuroanatomically ubiquitous. In mammals (including humans) cortical neurons become mature, post-mitotic cells early on during development (40), and maintain the stable post-mitotic state for decades (41). Further evidence suggests that the human neocortex does not acquire any additional neurons after birth (41), and that brain tumors are not derived from mature, TD neurons, but rather, from aberrant neural stem cells found in the areas of the brain with regenerative ability (38,42). Neuronal cell death also typically occurs when TD neurons are induced to re-enter the cell cycle, either experimentally or due to cell stress (38,43).
Following neural insult and injury, the terminal zones of severed neurons swell into dystrophic growth cones and have been shown to have some regenerative capabilities when in the appropriate environment(s) (44)(45)(46)(47). Neurons of the dorsal root ganglia, for instance, have axons in both the peripheral neural system (PNS) and the CNS; however, only those ending in the PNS have capacity for regeneration (48). The inability of CNS neurons to regenerate neural fibers is related to the environment of the adult CNS following injury (49). When a neuronal fiber is severed, axonal regrowth is initially inhibited by the presence of myelin-associated inhibitors in the glial environment [see (50,51) for overview]. These myelin-associated inhibitors are released by both intact oligodendrocytes and myelin structures that may have been damaged by injury (47,(52)(53)(54)(55). Identified inhibitors include: Nogo, myelin-associated glycoprotein (Mag), oligodendrocyte myelin glycoprotein (Omgp), ephrin B3 and transmembrane semaphoring 4D (Sema4D) (54,56).
Following CNS injury, microglia, oligodendrocyte precursors, meningeal cells, and astrocytes are also recruited to the site. Activation of local inflammatory processes and induction of lipid-collagen matrices lead to gliosis and formation of a glial scar, which provides a physical barrier and further inhibits regrowth of the axon and outgrowth of neurites beyond the lesion site (1,47,54). Moreover, the recruited astrocytes enter a reactive state, in which they up-regulate and release chondroitin sulfate proteoglycans (CSPGs), creating a chemical gradient at the site of injury that is inhibitory to regrowth (47,57,58). Additional evidence suggests that the glial scar may also provide stability to the site of injury in the CNS, preventing further cellular degeneration, isolating the inflammatory response, and repairing the blood-brain barrier at the injury site (59)(60)(61).
Thus, the adult nervous system is largely unable to regenerate following irreparable neuronal damage and/or loss (1,(62)(63)(64). This presents challenges (viz. a "therapeutic/recuperative problem") to developing effective interventions for neural insult, and is an increasing clinical (and socio-economic) concern as the incidence of neurological injury and global prevalence of neurodegenerative disease rise (1,6,38,47,54,65). structural, and cell-based approaches, either singularly or in combination. Although pharmacological approaches have demonstrated therapeutic benefit for management of symptoms and functional rehabilitation associated with SCI (66), TBI (67), and stroke (68,69), not all have been proven safe or effective in reparative or regenerative capacities in clinical application. Structural approaches, while perhaps limited in their reparative and regenerative capacities when employed in isolation, have demonstrated some success when employed in combination with cell-based approaches (70)(71)(72)(73)(74). We focus herein, however, on cell-based approaches to repair and possible regeneration of the injured CNS that have shown potential in preclinical and early clinical studies.
Cell-based therapies can have a variety (i.e., direct, indirect, or both types) of effects. Such therapies can be used to facilitate regeneration of neurons via implantation of specifically active cells in the adult CNS. Direct cell-based therapies generally involve use of implanted cells to replace or repair damaged neuronal and/or glial cells and tissue. Indirect effects involve use of implanted cells to contribute biomolecular and biochemical factors that modify the neural micro-and/or macroenvironment, providing trophic support that facilitates neural repair and regeneration (6,(75)(76)(77). Some cell-based therapies have both indirect and direct effects. Bone-marrow derived mesenchymal stem cells (BM-MSCs), for instance, exhibit immunosuppressive qualities and the ability to promote the proliferation of endogenous cells following stroke, in animal models (78)(79)(80)(81). These cells are also capable of trans-differentiation into various neural cell types, such as astrocytes, oligodendrocytes, and neurons, both in vitro and in vivo, and in in vivo animal models have been shown to demonstrate migratory capacity and actions in the CNS (82-92).

Stem Cells
Stem cell-based therapies for neural regeneration and repair garnered attention after the identification of specific regions of the adult human brain capable of maintaining the capacity for neuroregeneration throughout the human adult lifespan (6,77,(93)(94)(95).
Stem cell-based techniques have been increasingly innovative, with relatively rapid advances enabling the potential to combine stem-cell therapies with previously explored pharmacological, structural, and even other cell-based methods (96-99). For example, stem cells could be modified to deliver biomolecules or to replace damaged neurons, astrocytes, oligodendrocytes, etc. and thereby act directly and/or indirectly, as noted above (100).
As illustrated in Table 1, embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), neural stem/progenitor cells (NSCs), and induced pluripotent stem cells (iPSCs) have all been explored for use in cell therapies for neuroregeneration in a variety of models and applications.

Embryonic Stem Cells (ESCs), Fetal Stem Cells and Derivatives
ESCs were first cultured and isolated from mice (134), but can now be derived from donated human blastocysts following in-vitro fertilization (IVF) procedures (135,136), somatic cell nuclear transfer (137), human or mice fetal brains (120, 122), or existing hESC lines (there are currently 390 NIH-approved hESC and 70 unapproved cell lines 1 . ESCs are pluripotent and can proliferate almost indefinitely in vitro (135,138,254). Furthermore, ESCs have potential to differentiate into any cell type, including neurotransmitter or growth factor-secreting cells, neural stem cells (NSCs) and neural progenitor cells that can be further differentiated into neuronal subtypes, and/or glia (e.g., oligodendrocytes, astrocytes) capable of effecting roles in facilitating neural repair and/or regeneration (117,120,121,139,254,255).
Early preclinical studies employing in vivo mouse models demonstrated the ability of hESC-derived neural progenitor cells to integrate into host parenchyma, migrate along established pathways in the brain, and differentiate according to regionspecific cues (254). Various cell transplantation applications of hESC-derived, as well as mouse or human fetal-derived NSCs, in animal models of TBI suggest the potential of these cells to migrate to injured regions of the brain, differentiate into neurons and neuronal subtypes, and improve cognitive and motor functional recovery in the injured brain (121,122,139). Transplanted ESC-derived cells in ischemic animal models (e.g., rats subject to middle cerebral artery occlusion (MCAO)) have also demonstrated the ability to differentiate in vivo and to improve structural, functional, behavioral, and motor and sensory repair (123-125). NSCs and NPCs derived from ESCs have also been applied in preclinical animal models of stroke (126)(127)(128)(129)(130)(131) with marked improvements in the size of the infarct area, the level of differentiation into neurons and neuronal cell types post-transplantation, and improved behavioral deficits (256).
Transplanted ESC-derived NSCs have demonstrated functional and structural improvement in animal models of SCI, as well (101, 102, 104-107, 257,258). An ongoing phase I/II study (NCT02302157) is investigating the application of human ESC-derived oligodendrocyte progenitor cells (OPCs) in subjects with subacute cervical SCI; this clinical trial is supported, in part, by preclinical evidence of the safety and ability of these cells to promote neurite outgrowth in vitro and to facilitate myelination in vivo in rodent models of thoracic SCI (103).
Risks of inappropriate differentiation (i.e., teratoma and tumor formation), as well as technical issues arising from the possibility of immunological and graft rejection of the implanted tissues are major challenges that are still to be overcome (140)(141)(142). As well, ethical controversies surrounding the source of ESCs has led to alternative sources of pluripotent stem cells being explored (143,144). 2 Fetally-derived NSCs can be isolated from fetal brain tissue, with minute quantities also reported in bone marrow and umbilical cord blood (259). While these cells readily give rise to neurons, astrocytes, and oligodendrocytes, they are less versatile and proliferative than ESCs because they maintain some of the characteristics of the region of the brain from which they were derived 3 (100, 145). Transplantation of human fetal stem cells and their derivatives have shown promise in: increasing neuronal survival and decreasing lesion size in animal models of TBI through increased angiogenesis and reduced astrogliosis (122, 147); and, neuronal regrowth, functional and structural improvement following transplant in animal models of SCI (108-110). Clinical trials involving fetal stem cells or their derivatives for the treatment of SCI and stroke are completed and ongoing. A Phase I study is underway that is investigating the safety and efficacy of human fetal spinal cord-derived NSCs (NSI-566) for the treatment of chronic SCI (NCT01772810), and a Phase II study investigating the effect of human central nervous system stem cells (HuCNS-SCs) on patients with thoracic spinal cord injury has been completed (NCT01321333). Yet another Phase I/II clinical trial is ongoing that is evaluating the effect of NSI-566 cells on motor deficits in stroke patients (NCT03296618). Despite such progress in preclinical and clinical fields, ESC-and fetally-derived stem cells have given rise to ethical concerns regarding the source and process of their procurement, technical considerations focal to possible risks incurred during transplantation, and unregulated tissue growth in situ [for overview, see (260)].
The use of adult NSCs/NPCs (derived from biopsy tissue from brain or spinal cord) in cell therapies could, if validated in preclinical proof-of-concept studies, allow for autologous cell transplantation and repair by endogenous NSCs, thereby potentially minimizing risks of incompatibility. Early studies in animal models of SCI and TBI have suggested the potential viability of this approach (isolation, in vitro expansion, and transplantation), either alone or in combination with other methods (e.g., bone marrow-derived MSCs) for provision of neuroprotection, immunomodulation and facilitation of remyelination and functional recovery of the injured spinal cord (149-151, 153, 154, 156).
In transplantation-based therapy, NSCs or NPCs are implanted within defined areas of the CNS 1 . Transplanted NSCs and NPCs facilitate neuroregeneration and therapeutic plasticity in the injured CNS via processes of neuroprotection [i.e., the "bystander" "mechanism" in SCI, see (262) for overview; (132,155) for ischemic stroke], neurotrophic support (262); immunomodulation (164), and cell replacement and integration (111). Broadly speaking, neuroprotection is the activation of pathways that prevent further neuronal cell death, while neurogenesis involves proliferation and differentiation of endogenous neural stem and progenitor cells (259,262), or implanted NSCs capable of cell replacement. Lu et al. (113) found improved motor functionality following SCI in rodents, when rat-and human-derived NPCs, human ESC-derived NSCs, and human iPSC-derived NSCs were implanted at injury sites (112,114,115,263). Specifically, it was observed that these cells were able to extend multiple axons over long distances, and form synapses with host neurons (112, 264). Research suggests that endogenous NSCs and the immune system share secreted mediators (chemokines, cytokines) and receptors relevant both to inflammation and the maintenance, structure, and function of endogenous neural stem cell niches in the brain (165,166,265). Transplanted exogenous NSCs may modulate local immune responses and secrete growth factors that are conducive to neuronal growth (132,133). Indeed, both in vitro and in vivo proof-of-concept studies have demonstrated the potential of NSCs as a cell-based therapy for SCI (266)(267)(268), stroke (269), and TBI (266,268,269), based, at least in part, upon the capability for defined differentiation and target specific integration and functional activity of these cells when introduced at or proximate to sites of neural injury (259).
However, NSCs and NPCs from adult tissues cannot be easily engaged to generate certain types of neurons [e.g., dopamine or motor neurons; (162)] 2 , nor are they easily isolated or expanded from the regions of the human brain that contain NSCs [i.e., the subgranular zone of the hippocampus, and the subventricular zone of the striatum; (94)], in part, because they are present in rather low numbers (100). Moreover, despite the viability and potential value of transplanted NSCs as a therapeutic approach, gaps exist in knowledge about the activity and effect(s) of NSCs in vivo with regard to migration mechanisms and the optimum number of NSCs needed to facilitate regeneration in different types of lesions (166,259). The method and timing of delivery, and potential interactions between the transplanted NSCs and the host immune system may also impact the induction and extent of neuroregenerative effects produced by these cells (160,166).
The CNS environment at the site of neural injury in SCI, TBI, or stroke is not conducive to neuroregeneration (270). This is due to the activation of inhibitory pathways, glial scar formation, and the lack of guiding astrocytes essential for axonal regrowth (6). As a result, many applications of NSCs have resulted in poor cell survival, failed integration into host tissue, and poor, if not uncontrolled, differentiation of cells (6,153,271). Additionally, while NSC transplants derived from ESCs have shown increased risk for formation of tumor growth compared to NSCs that have been derived from either fetal or adult brain tissues, that is not to say that these cells do not also have associated risks of tumor formation 1 (100, 260). Furthermore, given that a source of NSCs is from ESCs and fetal tissues, research and potential therapies using these stem cells generate concerns about derivation, cost, supply, and accessibility of these cells (11,272,273). In sum, these challenges, as well as mixed evidence of safety and efficacy in animal studies (152,153,274), have hindered translation of adult brain-derived NSCs/NPCs into clinical therapies (100, 259,260).

Ongoing Trials
There are currently no stem cell therapies for neural injuries (namely, SCI, TBI, or stroke) that have received US FDA market approval for clinical application in humans, although there are 19 ongoing trials investigating stem cell therapies for SCI (as of May 2018; see Table 2 for details) 3 . Four of these are Phase 1, seven are Phase 1/2, six are Phase 2, one is phase 2/3, and one is unspecified. There are also 6 trials investigating stem cell therapies for TBI, and 22 trials investigating stem cell therapies for application to stroke 4 .
The majority (12)  Here we highlight several of these ongoing trials pertaining to stem cell therapies for SCI to demonstrate the types of studies being conducted. NCT02326662 (277) is a non-randomized interventional study assessing the safety and efficacy of MSCderived autologous NSC transplantation for individuals with traumatic SCI (NCT02326662). The aim is to recruit 30 participants (paraplegic and tetraplegic patients in the acute, subchronic, and chronic phases of SCI), who will receive transplants of autologous MSC-derived NSCs by intraspinal and intrathecal injection, along with RMx Biomatrix scaffolding as needed. NCT02688049 is a Phase 1/2 randomized, double-blind clinical trial assessing the efficacy and safety of NeuroRegen Scaffold TM when combined with MSCs or NSCs for chronic SCI repair. It is a two-arm study, with patients in one arm receiving the NeuroRegen Scaffold TM combined with MSC transplantation therapy following SCI, and those in the other arm receiving NeuroRegen Scaffold TM combined with NSC transplantation following SCI (NCT02688049). NCT01676441 is a Phase 2/3 clinical trial being conducted in the Republic of Korea, that aims to evaluate the safety and efficacy of bone marrowderived MSCs when transplanted into patients with chronic SCI. All participants (32) will receive MSC transplants directly to the injured spinal cord lesion site via laminectomy, and following recovery from the procedure, will undergo 4 weeks of physical and occupational therapy (NCT01676441). Participants will undergo magnetic resonance and diffusion tensor imaging and electromyography and nerve conduction testing at 6 months post-operatively, and be assessed for motor and sensory function as well as adverse events (using the American Spinal Injury Association (ASIA) scale) 12 months post-operatively (NCT01676441) 4 .

Reported Results
At this writing, 13 trails involving stem cell therapies for SCI on ClinicalTrials.gov have been completed, although the results from all of these studies are not yet available. Park et al. (294) reported long-term outcomes (i.e., changes in motor power grade of extremities, MRI, and electrophysiological recordings) of 3 SCI patients who received intramedullary transplantations of autologous bone marrow-derived MSCs (sourced from the iliac bone of patients). The patients were identified from an initial cohort of 10 patients as those who showed evidence of improvement in their activities of daily living (ADL) at 6 months following MSC-transplant (295). Patients had received direct injections of MSCs (8 × 10 6 ) to the spinal cord, in addition to introduction of MSCs (4 × 10 7 ) to the intradural space. At 4 and 8 weeks post-operatively, patients received additional injections of MSCs (5 × 10 7 ) via lumbar puncture. In this long-term follow-up, none of the 3 patients exhibited tumorigenesis or complications associated with the intramedullary injection of MSCs. Additionally, 2 of the 3 patients showed evidence of gradual improvement in motor power of the upper extremities, as well as evidence of the disappearance of cavity margins, which may suggest the ability of BM-derived MSCs to diminish glial scarring (294). The results of this study do not contradict those of previous studies [e.g., (296,297); see (6) for an overview of completed trials involving stem cells for SCI], which similarly found slight motor improvements without significant complications associated with MSC transplantation in humans with SCI (294).
The results of such studies suggest that stem cell therapies can be especially beneficial for functional recovery in the acute and subacute stages of SCI, but that improvements incurred during the later and chronic stages of SCI are characteristically less significant (6).
Clinical trials involving stem cell therapies for TBI and stroke have also been completed. Results for MSC transplants in TBI patients have been promising, with both (298) and (299) reporting enhancement of neurological function and recovery among adults and pediatric patients following injury. Kalladka et al. (300) recently reported on the use of human fetal brainderived NSCs (CTX0E03) in patients with chronic ischemic stroke in a phase 1 study. This single-site, dose-escalation study found improved neurological function and no immunological or cell transplant-related adverse events in a sample of 13 men over the age of 60 who received intracerebral implants of (2, 5, 10, or 20 million) NSCs, up to 2 years post-transplant (300).

MORE RECENT APPROACHES: IPSCS AND REPROGRAMMED AUTOLOGOUS CELLS
Alternatives to ESC and fetally-derived cell-based transplantation therapies received renewed attention upon the discovery that adult somatic cells could be induced to become pluripotent stem cells (190). Adult somatic cells include MSC derived from bone marrow, adipose, or epidermal tissues (301,302). NSCs may also be induced from amniotic fluid stem cells (193). Induced pluripotent stem cells (iPSCs) have shown some promise in their ability to generate patient-specific, autologous pluripotent cells and tissues, although their ability to retain all autologous characteristics has recently come into question (194,195). While iPSCs have been predominantly been used in animal and in vitro models, these studies have both significantly contributed to an understanding of neurodegenerative diseases, and are being viewed as significant resource tools for discovering novel therapeutic strategies against neurodegenerative disease and particular types of neurological injury (144,303).
iPSCs Researchers successfully demonstrated that induced pluripotent stem cells (iPSCs) could be generated from mouse embryonic fibroblasts (190) and from adult fibroblasts (189,191,192,199) that are cultured in the presence of some combination of four transcription factors (Oct4, Sox2, Klf2, and c-Myc; or Oct4, Sox2, Nanog, and Lin28)-rather than from embryonically-or fetallyderived cells (11,144,190,191). Since then, iPSCs have become an attractive source of pluripotent stem cells for use in neural replacement and regeneration therapies, given their potential to generate a "virtually limitless supply" of autologous iPSCsthus resolving ethical concerns regarding the source of stem cells and risks associated with immunological rejection in transplant recipients (11,196). In neural therapies, iPSCs and their progeny can facilitate regeneration of neurons, induce axonal remyelination, provide trophic support and immunomodulation, and modify the extracellular microenvironment (105, 167,177,187).

Reprogramming and Differentiation of iPSCs for Neural Insult
The first step in generating iPSCs is to select an appropriate type of donor cell (201). Currently, the majority (i.e., 80%) of published studies of cellular reprogramming employ fibroblasts in that they are easy to obtain, purify, maintain, and have significant potential for autologous transplantation (11,191,273). Other somatic donor cells include: melanocytes, adipocytes, CD34+ cells, mesenchymal stem cells, human primary keratinocytes, umbilical cord blood CD133+ cells, and peripheral blood mononuclear cells (199,208,273,(304)(305)(306)(307).
Each of these cell types has a characteristic differentiation profile related to its epigenetic memory (201,308,309). Such "memory" (e.g., particular patterns of DNA methylation, histone acetylation and phosphorylation) can influence patterns of expression and differentiation profiles of the resultant pluripotent stem cells (201,309). Cells of different origins can also differ in their reprogramming efficiencies (11,310). When designing cell therapies for neurotrauma, it is important to consider the donor cell source in light of the ease of isolation/expansion, the differentiation profile, the pathology of the CNS injury of interest, and the intended destination of the cell therapy in the injured patient.
Skin fibroblasts, for instance, are easily obtained but can require more time, resources, and manipulation to generate iPSCs due to their low programming efficiency (311,312). Given the increased time required for reprogramming, they also have potential for increased mutations in vitro. Keratinocytes and melanocytes show therapeutic potential as sources of iPSCs: both are relatively easy to obtain from skin biopsies or plucked hairs, have relatively short reprogramming periods and high reprogramming efficiencies (203). Some research suggests the potential for these cells to be reprogrammed to neural stem cells, though further research is needed to improve the reprogramming process and validate its potential for differentiation and neuroregenerative applications (11,313). Use of CD133+ cells from umbilical cord blood has been explored due to these cells 'relatively high reprogramming efficiency and pluripotency (205); however, their use in therapies for CNS insult remains questionable, as there remains a need to characterize their NSC differentiation potential and optimal culture conditions (11,314). CD34+ cells, derived from bone marrow and umbilical cord blood, are not recommended for clinical use due to their low availability and low reprogramming efficiency (206,207). Stem cells derived from adipose tissue show notable promise as a reliable source of iPSCs: they are easily obtained during liposuction procedures, require fewer genomic changes to generate iPSCs, and the reprogramming period is both relatively short and efficient (208,315).
Since the publication of studies by (189), a number of protocols for the development of iPSCs have become available that use these various adult-derived donor cells (some autologous) and employ different combinations of transcription factors (TFs) and delivery methods-with the goal of finding a protocol that optimally balances efficiency, safety and related risks (201,(311)(312)(313)(314)(315)(316)(317)(318)(319)(320)(321). Some TF delivery methods involve the use of viruses, such as lentiviruses, to integrate viral DNA into replicating regions of the host genome (322). The use of viral vectors to deliver reprogramming factors can, however, lead to chromosomal disruption, expression of transgenes, and/or mutations (322). More recent "scarless" technologies have been introduced that use episomal vectors, synthetic mRNAs and Sendai viruses that do not integrate to the genome during reprogramming of adult somatic cells into iPSCs (198,(323)(324)(325). Kim et al. (199) developed a vector-free method to generate human iPSCs directly from human fibroblasts through delivery of reprogramming proteins (Oct4, Sox2, Klf4, and c-Myc) fused to cell penetrating peptides capable of crossing the cell membrane (199). These "scarless" and vector-free systems reduce potential risks of chromosomal disruption associated with DNA transfection and genome manipulation.
Studies in animal models of SCI that involve transplantation of human NSCs derived from iPSCs also suggest that difficulties remain in ensuring in vivo differentiation of cells into appropriate neural cell lineages and with tumorigenicity-both of which may hinder successful cell engraftment and functional recovery (326,327). Evidence suggests that the local CNS microenvironment and pro-inflammatory factors (and associated neuroinflammation) can influence transplanted cell engraftment, differentiation and survival (326). In light of this, optimization of protocols for human cell reprogramming and the generation of iPSCs for use in clinical trials remains a work in progress in an attempt to achieve balance in safety and efficiency when applied in humans for certain types of neurotrauma (11,144,328).
Direct reprogramming via chromatin remodeling involves opening the chromatin in somatic cells, and facilitating sustained exposure to a set of transcription factors that effectively converts the somatic cells to NSC/NPCs; chromatin remodeling enables NSCs/NPCs to maintain their new identities and bypass the intermediate pluripotent state (337,344,345). For direct reprogramming approaches, bypassing the intermediate pluripotent state reduces the associated reprogramming risks of genetic and epigenetic abnormalities (346,347). Continuing research is further exploring those combinations of TFs that could be most effective in direct reprogramming somatic cells into neuronal subtypes (e.g., dopaminergic, GABA-ergic, glutamatergic, and motor neurons), as well as distinct types of glial cells [i.e., oligodendrocytes, astrocytes, and Schwann cells; (325,348)].

POSSIBLE APPLICATIONS OF AUTOLOGOUS IPSC-DERIVED CELL TYPES AND DRNPCS
NPCs have potential to participate in and facilitate central neural regeneration and repair by functioning to replace lost or damaged neurons, prompt axonal remyelination, and provide local trophic support (111,132,155,262). Autologous iPSC-NPCs could be generated using a patient's somatic cells, and reprogrammed via dual SMAD inhibition (209,349), embryoid body formation and differentiation into neural rosettes (210), or via the piggybac transposon system paired with induction of the NOTCH pathway (211,324). Dimos et al. (350) generated autologous iPSCs from patient-specific fibroblasts that were then differentiated into motor neurons. As previously mentioned, drNPCs may also be generated via direct reprogramming of autologous somatic cells. In mouse models of SCI, transplanted iPSC-NPCs induced axonal remyelination and regeneration, inhibition of (further) cell death, and improvements in a number of behavioral outcomes (169,170,172). These cells have also shown the ability to differentiate in vivo into neurons and glial cells, migrate (169), and effectively integrate to host tissue within the CNS (114).

Optimization of iPSC Production and Differentiation
Several important questions remain regarding reprogramming of human cells that must be considered pursuant to their use in clinical applications. For example, it will be important to discern to what extent iPSCs retain epigenetic memory of their donor cell types, and whether the induced pluripotency is equivalent to that seen in ESCs (144,309,351). It is also necessary to determine the extent to which different reprogramming methods (choice of donor cell and iPSC line, cell selection during propagation, and culture conditions) influences genetic variability and functionality of the differentiated cells (144,310). While genetic variability between iPSC lines could theoretically be resolved via gene editing, human pluripotent stem cells have demonstrated resilience to conventional gene targeting methods, and even those methods that have worked (352)(353)(354) have been shown to be both inefficient and time consuming (144). Advancements made in the use of novel gene-editing systems, such as CRISPR/Cas-9 and its variants, may offer a possible solution to this demonstrated resilience in iPSCs to targeted gene-editing (144,355).

Mutations, Tumorigenicity, and Teratoma Formation
Concerns regarding mutations and tumorigencitiy are focal to any consideration of the development and use of reprogrammed, differentiated iPSCs. iPSCs can acquire mutations during both the reprogramming process and in vitro expansion; furthermore, some protocols for generation of iPSCs call for the use of c-Myc-a proto-oncogene associated with various types of cancers (325,356,357). Risk of tumorigenicity and accumulation of genetic alterations might be reduced by selecting appropriate cell source(s) for the generation of iPSCs (i.e. adipose cells, keratinocytes, or melanocytes) (11,310). Direct reprogramming may also offer a way to reduce these risks, as reprogrammed cells do not require an intermediate pluripotent stage (329)(330)(331)(332).
Yet, even with sufficient controls for mutation during these phases of development, there remains a definitive need for an efficient technique for evaluating differentiation and screening for mutagenesis and potential tumor-and/or teratoma formation both prior to and following transplantation. Antibodies that target high-risk cell subpopulations and selective in vivo ablation of transplanted cells are potential solutions being explored to reduce this risk (358), and further studies are needed to more fully define the effectiveness of this, and other methods of insuring stability of these cells.

Immunological Interactions and Possible Rejection
A growing body of literature describes the numerous pathways of communication that exist between the immune system and the CNS-namely, the role that such communication has in the CNS' ability to maintain homeostasis and to react to neurological injury and trauma [e.g., neuroinflammation, (359)(360)(361)(362)]. Communication between these two vital systems is mediated primarily through immune regulatory molecules that are released by cells residing in the CNS, such as microglia, mast cells, astrocytes, and oligodendrocytes (361)(362)(363)(364)(365).
Following neurotrauma, the CNS undergoes pathophysiological changes that both affect and are affected by the innate and adaptive arms of the immune system. After acute stroke, for instance, brain ischaemia, damage to tissue and subsequent release of pro-inflammatory factors (e.g., matrix metalloproteinase-3 (MMP-3), ATP, alpha-synuclein, and neuromelanin, (366,367)) can activate innate immune (via leukocytes, toll-like receptors, and the lectin pathway) and neuroinflammatory responses, such as the release of cytokines and activation of resident microglial cells (359,365,368). T and B cells mediate the adaptive immune responses to neurological damage, providing both potentially protective and potentially damaging autoimmune/reactive effects on neural tissue, if inflammation is prolonged and unregulated [ (362,365,(369)(370)(371); for a thorough overview of the innate and adaptive immune responses to neurotrauma, see (359,371)].
For cell therapies intended to treat the effects of neurotrauma, neuroinflammation and the potential for an immune response must be considered. In some cases, the administration of antibiotics, immunosuppresants, or other small-molecule antiinflammatory agents [e.g., N-palmitoylethanolamine (PEA)] may be required, particularly following or alongside transplant or injection of tissue/cells into injured patients (362,372,373). However, one must also consider the effect that administration of such immunosuppressants may have on the stem cell therapies, cell differentiation and cell engraftment, as well as the optimum time for delivery of these substances (374).
In theory, the use of autologous adult somatic cells to generate iPSCs and progeny would avoid or at least significantly reduce immunogenicity in stem cell transplant therapies. However, Zhao et al. (375) suggested that transplantable stem cells could be subject to aberrant DNA methylation during the reprogramming process, and that such changes to the DNA might trigger an immune response after transplantation in the host. Other potential sources of immunogenicity may include culture reagents, protein expression, maturity of transplanted cells, or factors of the micro-or macro-environment at the site of transplantation (376). Others have explored the potential immunogenicity of autologous iPSCs using mouse (377,378) and primate models (379), and these studies appear to contradict the results reported by Zhao et al. (375). Such ambiguity establishes the need to more stringently assess potential sources of immunogenicity in iPSC-derived cell therapies prior to their clinical use.

Safety and Use: Addressing Ethical, Legal and Social Issues
Interest in stem cell research is sustained by the potential contribution that these cells and their progeny can make to transplant-based therapies for neurological repair and regeneration (260,380,381). Indeed, research suggests that stem cell transplantation affords potential as a therapeutic strategy for central neural injury and disease (260,382,383). Initially focused on use of ESCs as sources of pluripotent stem cells, the field was soon rife with ethical debate related to controversies surrounding the sources of ESCs, implications relevant to the status of early-stage human embryos (380), limited supply, high cost, and associated risks of tumorigenicity and immune rejection after transplantation.
In light of this, more recent basic and preclinical studies of regenerative therapies for CNS insult have shifted toward use of iPSCs and their derivatives that are generated from adult somatic cells. As noted above, iPSC-derived and directly reprogrammed neuronal and glial cell types can be generated from cell sources that are not (or at least less) controversial and more readily available. Such cell sources can be autologousderived from tissue samples directly from patients-and thus, can eliminate or significantly reduce risks of immunological rejection after transplantation, and may appeal to current incentives for personalized and precision medicine. As well, developments have been made in reducing time spent in vitro during the reprogramming processes, improving selection of cell type, and increasing efficiency of methods to remove harmful cells preand post-transplantation, which, when taken together, can reduce risks of tumorigencity in iPSC transplant-based therapies.
To be sure, autologous reprogrammed cells have a number of attributes that make them attractive and potentially viable alternatives (to ESCs and fetal stem cells) in cellbased therapeutic approaches for particular types of CNS injury and disease. Yet, while this approach has potential to fulfill therapeutic goals of enabling partial to complete recovery of defined functions, ethico-legal and social issues persist. As described in detail elsewhere (260,384,385) such issues involve the unknowns of intermediate and long-term use of new techniques and technologies in practice. For example, (1) how such unknowns impact the validity and contingencies of informed consent; (2) the need for continuity of research and clinical care (to address emerging issues consequential to sustained use-in-practice); (3) legal liabilities and process of claims if/when adverse events occur; and (4) provision/distribution of resources and services (to furnish cells, as well clinical interventions, and ongoing support) in and across various populations in need.
We have proposed a risk assessment approach to more specifically define and address the issues, questions and problems generated by possible uses of emerging neuroscientific and neurotechnological developments (386,387); but address does not assure resolution. While some concerns relate to safety and effectiveness (e.g., possibilities of unanticipated consequences, runaway effects) with regard for patients' welfare (i.e., via integrity of informed consent and availability of sustainable clinical care), others reflect recognition of problematic distribution of medical goods [for overview, see (388)]. Both safety and distribution can be met, to some extent, by rigorous quality control and expediency in the manufacture and delivery of cells. Still, it remains to be determined if the type and extent of broad-based medical care (i.e., trained personnel and institutional resources) can and/or will be made available to perform the procedures and insure sustained post-implantation evaluations and care.
We have opined, and re-iterate here, that any consideration of the ethics of biomedicine must regard economic factors, and in many cases, economics of biomedical research and care are determined by policy and law (389,390). Here discussion of the viability and value of NSCs-or any biomedical technique and/or technology-centers upon the interactive roles of regulatory policy in establishing both standards of care and fiscal (i.e., insurance) subsidy of these approaches in patient care. Suffice it to note that the gravitas-and international relevance and importance-of this situation is such that a complete discussion of economic and policy issues focal to NSCs or translational neuroscience would be significant, and is therefore beyond the scope of this paper.

CONCLUSIONS AND FUTURE VISTAS
Research and possible use of stem cell therapies toward mitigating the effects of neurotrauma, and inducing neurological repair and regeneration are gaining momentum and attention-in both the professional and public spheres. Emerging techniques and technologies of cell development, manufacture and delivery are progressing at a considerable pace. A number of previously contentious technical and ethical issues and questions have been met and resolved, at least in part, by the advent of ever newer forms of stem cells. Of these, autologous stem cells appear to offer a number of advantages (e.g., immunological compatibility and lack of rejection, personalized therapy, non-fetal, or embryonic derivation) that would suggest their value in and for clinical use in effecting neurological repair and regeneration (following neurotrauma and in treatment of certain neurological diseases). Yet, it is likely that there will not be a single stem cell therapy for CNS injury, but instead a more customizable (pathology-and patient-specific) approach.
Regardless of iteration or approach, it will be important and necessary to develop and insure high quality, secure and rapid throughput manufacturing processes that enable time-and costefficient delivery of these cells. But manufacture is but one cog in a multi-spoked wheel of biomedical research and care. As noted, if developments in the brain sciences are to be translated into safe, affordable and accessible clinical care that maintain high quality, then medicine, as well as economics, regulatory policy and law must be aligned in order to support and sustain meaningful benefit to patients in need. Simply put, the prevalence and multi-dimensional burden of neurological injuries and neurodegenerative diseases demand the further exploration and pursuit of NSCs-and other therapeutic strategies-that are both promising and feasible.