Frontiers in Stem Cell and Regenerative Medicine Research: Volume 9 E-Book
106,56 €
Mehr erfahren.
- Herausgeber: Bentham Science Publishers
- Kategorie: Wissenschaft und neue Technologien
- Serie: Frontiers in Stem Cell and Regenerative Medicine Research
- Sprache: Englisch
Stem cell and regenerative medicine research is an important area of clinical research which promises to change the face of medicine as it will be practiced in the years to come. Challenges in the 21st century to combat diseases such as cancer, Alzheimer’s disease and retinal disorders, among others, may well be addressed employing stem cell therapies and tissue regeneration techniques. Frontiers in Stem Cell and Regenerative Medicine Research is essential reading for researchers seeking updates in stem cell therapeutics and regenerative medicine.
This volume includes current literature on a variety of topics:
-the utility of exogenous and endogenous neural stem cells in spinal cord injury
-somatic cells for human induced pluripotent stem cells (iPSCs)
-reactive oxygen species (ROS) mediated cellular signaling for stem cell differentiation
-the therapeutic potential of microRNAs in cardiac diseases
-stem cell therapy for the treatment of malaria
Sie lesen das E-Book in den Legimi-Apps auf:
Seitenzahl: 468
Veröffentlichungsjahr: 2020
Ähnliche
BENTHAM SCIENCE PUBLISHERS LTD.
End User License Agreement (for non-institutional, personal use)
This is an agreement between you and Bentham Science Publishers Ltd. Please read this License Agreement carefully before using the book/echapter/ejournal (“Work”). Your use of the Work constitutes your agreement to the terms and conditions set forth in this License Agreement. If you do not agree to these terms and conditions then you should not use the Work.
Bentham Science Publishers agrees to grant you a non-exclusive, non-transferable limited license to use the Work subject to and in accordance with the following terms and conditions. This License Agreement is for non-library, personal use only. For a library / institutional / multi user license in respect of the Work, please contact: [email protected].
Usage Rules:
All rights reserved: The Work is the subject of copyright and Bentham Science Publishers either owns the Work (and the copyright in it) or is licensed to distribute the Work. You shall not copy, reproduce, modify, remove, delete, augment, add to, publish, transmit, sell, resell, create derivative works from, or in any way exploit the Work or make the Work available for others to do any of the same, in any form or by any means, in whole or in part, in each case without the prior written permission of Bentham Science Publishers, unless stated otherwise in this License Agreement.You may download a copy of the Work on one occasion to one personal computer (including tablet, laptop, desktop, or other such devices). You may make one back-up copy of the Work to avoid losing it.The unauthorised use or distribution of copyrighted or other proprietary content is illegal and could subject you to liability for substantial money damages. You will be liable for any damage resulting from your misuse of the Work or any violation of this License Agreement, including any infringement by you of copyrights or proprietary rights.Disclaimer:
Bentham Science Publishers does not guarantee that the information in the Work is error-free, or warrant that it will meet your requirements or that access to the Work will be uninterrupted or error-free. The Work is provided "as is" without warranty of any kind, either express or implied or statutory, including, without limitation, implied warranties of merchantability and fitness for a particular purpose. The entire risk as to the results and performance of the Work is assumed by you. No responsibility is assumed by Bentham Science Publishers, its staff, editors and/or authors for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products instruction, advertisements or ideas contained in the Work.
Limitation of Liability:
In no event will Bentham Science Publishers, its staff, editors and/or authors, be liable for any damages, including, without limitation, special, incidental and/or consequential damages and/or damages for lost data and/or profits arising out of (whether directly or indirectly) the use or inability to use the Work. The entire liability of Bentham Science Publishers shall be limited to the amount actually paid by you for the Work.
General:
Any dispute or claim arising out of or in connection with this License Agreement or the Work (including non-contractual disputes or claims) will be governed by and construed in accordance with the laws of Singapore. Each party agrees that the courts of the state of Singapore shall have exclusive jurisdiction to settle any dispute or claim arising out of or in connection with this License Agreement or the Work (including non-contractual disputes or claims).Your rights under this License Agreement will automatically terminate without notice and without the need for a court order if at any point you breach any terms of this License Agreement. In no event will any delay or failure by Bentham Science Publishers in enforcing your compliance with this License Agreement constitute a waiver of any of its rights.You acknowledge that you have read this License Agreement, and agree to be bound by its terms and conditions. To the extent that any other terms and conditions presented on any website of Bentham Science Publishers conflict with, or are inconsistent with, the terms and conditions set out in this License Agreement, you acknowledge that the terms and conditions set out in this License Agreement shall prevail.Bentham Science Publishers Pte. Ltd. 80 Robinson Road #02-00 Singapore 068898 Singapore Email: [email protected]
PREFACE
The ninth volume of ‘Frontiers in Stem Cell and Regenerative Medicine Research’ presents important recent developments in this fast growing field.
Hasan and Wu review the current literature on the utility of exogenous and endogenous neural stem cells in spinal cord injury. Miguel et al. focus on the various sources of somatic cells for human induced pluripotent stem cells (iPSC) generation, their characterization, and the progress on directed differentiation toward several cell types.
Cell signaling and redox reactions are involved in the regulation of neural-lineage cells for reprograming of adipose-derived stem cells. Abrahamse et al. discuss the role of reactive oxygen species (ROS) and ROS mediated cellular signaling for differentiation of stem cells.
The human skin represents the largest and most accessible part of the body that is exposed to infections, physical wounds, diseases etc. Titorencu et al. describe commercially available skin substitutes that are in demand and also described the use of stem cells for skin regeneration. Recently, the role of microRNA in cardiac conduction diseases, arrhythmogenesis and their therapeutic potential has caught the attention of researchers. Sankaranarayanan et al. have reviewed the role of miRNAs in cardiac rhythmic disorders and their potential in diagnosis and treatment. Finally, Rivera et al. discuss the use of stem cell therapy in the treatment of malaria.
We owe our special thanks to all the contributors for their valuable contribution in bringing together the ninth volume of this book series. We also thank the editorial staff of Bentham Science Publishers for their help and support.
List of Contributors
Insights into Exogenous, Endogenous and Combination Therapies of Neural Stem Cells in Spinal Cord Injury
Hunaid Hasan1,*,Ping Wu2Abstract
Advancements in the understanding of spinal cord injury (SCI) and repair have taken great leaps in the past decade. Although understanding has evolved significantly there continues to be major limitations in the clinical interventions available for patients with spinal cord injuries. Exogenous stem cells (ExNSC) have progressed to human clinical studies, but have limitations due to ethical issues, technique and long term outcomes. However, the use of ExSCs through a stimulatory effect on growth factors, cytokine production and neurotrophic factors post injury may be beneficial. Bone marrow derived stem cells, mesenchymal stem cells, embryonic stem cells, umbilical cord stem cells, adipose derived stem cells, NSCs, Schwann cells grafts and olfactory ensheathing cells have been various types of exogenous cell types and techniques used in SCIs. The role of endogenous stem cells (eNSCs) in SCI has been promising, but still requires better lineage analyses to fully understand the responses of NSCs after SCI. It has been demonstrated that there exists a bidirectional interaction within the neuro vascular system forming the neuro vascular niche. Purinergic receptor activation was found to alter the intrinsic properties of the ependymal stem/progenitor cells enhancing regulation of proliferation, differentiation and lineage specification after a SCI. Therapies have been described using nervous tissue in combination with various synthetic bridges to overcome the structural barriers of regeneration through bypassing the injured area. More recently, newer techniques such as electrical stimulation have been described to stimulate mature neuronal differentiation. Various groups have emphasized that the glial scar is counter productive. Anderson et al. have shown the beneficial effects of a chronic glial scar in neural tissue preservation after SCI. Moreover, they have demonstrated higher levels of chondroitin sulfate proteoglycans in injured spinal cords independent of the glial scar. In sum, we have reviewed the previous and current literature on NSC and SCI to address the neurobiological utility of NSCs in spinal cord injury.
INTRODUCTION
Advancements in the understanding of spinal cord injury (SCI) and repair have taken great leaps in the past decade. The greatest challenge in SCI research has been the translation of what is known at the basic science level, and applying it to the clinical setting. Although, the understanding of the molecular basis of SCI has become more cogent, the therapeutic options continue to be limited. Although neural stem cells (NSCs) have been studied for over a decade, they continue to be at a translational research stage. Initially, exogenous stem cells (ExSCs) were an exciting breakthrough. However due to various reasons discussed below their clinical application as independent interventions has started to diminish. Recently, endogenous stem cells (eNSCs) have become more of an interest in SCI. Nature's endogenous process of healing spinal cord injuries was once thought to be counterproductive. However, as discussed below there has been evidence to suggest on the contrary. In sum, the function of eNSCs has been encouraging, and could help intervene in altering this natural process of healing. Further, its translation into clinical practice could improve the quality of life for numerous patients affected by SCIs.
SPINAL CORD INJURY
Epidemiology
Epidemiologically, SCI has been and continues to be a huge burden to society. Some reports find the incidence to be as low as 8 and as high as 246 cases per million depending on political, geographical, sociological and economic differences [1-4]. The mean age has increased from 28.3 years in the 1970s to 37.1 years surveyed in 2005-2008. Most concerning has been the epidemiological age group affected. As the majority affected have been in their peak productive years of their lives [5]. Also, more than 50% of SCIs affect the cervical region of the spinal cord, which is more likely to cause total body paralysis [6-8].
Mechanism of SCIs were either traumatic or nontraumatic [3]. Traumatic causes in young adults (age 15-29 years) were motor vehicle accidents (50%), violence (12%) and sport-related injuries (10%) [7, 9, 10]. Traumatic causes in the elderly (age >65 years) were mainly accidental falls [8-12]. Non-traumatic causes in both age groups were congenital and inflammatory spinal cord disorders, tumor compression, vascular ischemia vertebral spondylosis [13].
Pathophysiology
Primary Phase
SCI has been categorized into primary and secondary phases [14, 15]. The primary phase occurs due to compression and contusive injury resulting in shearing, laceration and stretching of the spinal cord. This exerts forces causing disruption of axonal function [16, 17]. It was suggested that at this stage full transection of the spinal cord was rare. There was sparing of demyelinated axons found at the subpial rim [17-21]. Animal models suggest that a minimum of 10% preservation of original axons could lead to significant neurological recovery [22, 23].
Secondary Phase
The pathophysiology of the secondary phase injury was directly proportional to the injuries occurring in the primary injury phase [17]. This phase was marked by a negative environment for neuronal regeneration [17]. The pathophysiology was marked by various processes such as ischemia, excitotoxicity, vascular dysfunction, oxidative stress and inflammation resulting in cell death [18, 24, 25]. Secondary phase injury was deleterious to surviving surrounding neurons, which further impaired functional recovery [14, 26]. The secondary phase had sub-phases which were: immediate (<2 hours), acute (2 hours – 2 days), subacute (3 days to 2 weeks), intermediate (2-3 weeks – 6 months) and chronic (>6 months) [17, 18, 27, 28].
Immediate phase consisted of upregulation of tumor necrosis factor (TNF)-alpha, interleukin (IL)-beta and elevation of extracellular glutamate [29-32]. This resulted in necrosis of neurons secondary to ischemia, lipid peroxidation, hemorrhage, reactive oxygen species (ROS) production, edema and cell membrane disruption. This lead to early loss in function and neurogenic shock [16, 17, 23, 33, 34].
A patient with SCI has shown to be initially clinically intervened in the acute phase. This phase was further divided into early acute and subacute stages [17]. Genes that change after injury have been classified as either “early” or “late” genes. They were responsible for activating cascades at various stages [35]. After an SCI event there was an upregulation of pro-apoptotic, cell cycle and oxidative stress mediating genes. Moreover, there was downregulation of antiapoptotic genes, genes involved in neural excitation, neurotransmission, electrochemical gradient and preservation of the neuronal cytoskeleton. Both necrosis and apoptosis were processes of cell death after SCI [36-38]. Both neuronal and oligodendrocyte cell death were mediated through activation of the Fas receptors and p75 receptor signaling pathways [39-44]. Fas-mediated apoptosis caused demyelination and Wallerian degeneration [36, 39, 40, 45-48]. Whereas, Fas receptors in the spinal cord were present on oligodendrocytes, activated microglia and lymphocytes [40, 41, 49]. Therefore, blocking of Fas related mechanisms could have clinical implications [38, 39, 50].
Acute Stage
Early acute stage was marked by ischemia due to disruption of the microvasculature, hypotension and elevated interstitial pressures. This lead to decreased perfusion of the spinal cord after injury [33, 51-53] (Refer to Fig. (1)). The mechanism of secondary injury was likely due to direct mechanical disruption and the effect of inflammatory mediators leading to increased permeability of the blood-brain barrier and blood-spinal cord barrier [18, 54]. As this leakiness allowed an immune cascade to occur. This permeability window could potentially be a therapeutic window for intervention [17]. Since this process had both a neuroprotective and detrimental neurobiological effect it was described as a "double-edged sword". Initially, surrounding microglia proliferated and attracted peripheral leukocytes, which upregulated chemokine production [31, 55, 56]. Neutrophil migration helped in activation of glia and the respiratory burst [56, 57]. In the subacute phase after 72 hours, monocytes differentiated into macrophages and expressed mediators worsening secondary injury [58]. Microglia proliferation continued to persist for weeks [31]. Redox imbalance due to ROS and nitric oxide (NO) species, genes - HspBl and HspA4 were found to be important in the pathophysiology of SCI [16]. Microglia and macrophages produced both species [59-62]. Decreasing NO production was neuroprotective [59-62]. Moreover, peroxynitrite radical formation, a product of NO and superoxide were involved in neuronal apoptosis [63]. Calcium regulation was crucial in preventing cell death [64, 65]. Its dysregulation was responsible for production of free radicals and activation of calpains [64, 65]. Excitotoxic cell death has been postulated to be due to disruption of osmotic balance [66-68]. This cascade was due to disruption of active cell membrane function maintaining glutamate homeostasis [69]. This process leads to overstimulation of glutamate receptors and increased influx of sodium and calcium via the N-Methyl-D-aspartate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors [66-68]. Subacute phase was marked by the proliferation and hypertrophy of astrocytes with formation of the glial scar releasing inhibitory substances like chondroitin sulfate proteoglycans [18, 70-75] (Refer to Fig. (4)).
Fig. (1)) Spinal cord injury pathophysiology. This figure demonstrated the acute, subacute and chronic phases of SCI. As a result of primary and secondary injury mechanisms there has been a presence of edema, hemorrhage, inflammation, apoptosis, necrosis, excitotoxicity, lipid peroxidation, electrolyte imbalance, ischemia/vasospasm and blood vessel occlusion. Axonal demyelination and disruption of synaptic transmission occurred secondary to oligodendrocyte and neuronal death. Fluid-filled lentiform-shaped cavities or cysts in the center of the cord with surrounding hypertrophic astrocytes and macrophages in the subacute and chronic phases were also depicted. J Clin Invest. 2012 Nov 1; 122(11):3824-3834; Copyright © 2012, American Society for Clinical InvestigationIntermediate Phase
Intermediate phase was marked by maturation of the glial scar (Refer to Fig. (4)). Although not clinically significant, there was axonal sprouting of the corticospinal and reticulospinal tract fibers [16, 76].
Chronic Phase
Chronic phase was marked by further stabilization of the glial scar and syrinx development [18, 77] (Refer to Fig. (4)). The pathophysiology consisted of clearance of debris of neural tissue by microglia and macrophages with some sparing at the syrinx rim [28, 78, 79]. This stage negatively affected neuroregeneration [80].
CLINICAL MANAGEMENT
Management for SCI has been discussed along the surgical, medical and NSC axis.
Surgical
Early surgical decompression, less than 24 hours post injury has constantly been shown to improve neurological recovery through various trials such as the Surgical Timing in Acute Spinal Cord Injury study (STASCIS). Neurosurgical practices at this time used for SCIs have been notably more empirical than evidence based [81].
Medical
Medical management has been discussed in terms of supportive therapy, pharmacotherapy and rehabilitation.
Supportive
Supportive medical management depended on whether the injury involved the upper spinal cord or the lower spinal cord. Injury to the upper spinal cord lead to hypotension due to disruption of the central supraspinal sympathetic control [4, 82]. Minimum arterial pressures of 85 mmHg should be maintained to prevent spinal cord hypoperfusion [83]. First line management in SCI above the sixth thoracic level involved administrating intravenous crystalloids fluids followed by colloid fluids and vasopressors with inotropic, chronotropic and vasoconstrictive properties [84, 85]. Management of lower spinal cord injuries involved administrating vasopressors with only peripheral vasoconstriction [84, 86]. Both
types of SCI required subcutaneous heparin prophylaxis for venous thrombo-embolism for three months [4, 87].
Pharmacotherapy
Pharmacotherapy consisted of methylprednisolone sodium succinate (MPSS) [17]. MPSS has shown to have an immunomodulatory effect by reducing peroxidation of membranes and reducing inflammation [88]. National Acute Spinal Cord Injury Study (NASCIS) I, II, III have established dosage, timing and adverse effects of MPSS. 30mg/kg bolus with 5.4mg/kg/h for 23 hours was thought to be neuroprotective as per NASCIS II [89]. Optimal timing for administration of MPSS was between 3-8 hours. As per NASCIS III there was some additional motor improvement if given for 48 hours [89-91]. Adverse effects included increased risk of infections such as pneumonia [92, 93]. STASCIS showed that early decompression and MPSS had a synergistic effect in cervical cord injury [81, 93].
Exercise Rehabilitation
Exercise rehabilitation was complementary to the medical and surgical strategies [17]. Studies have shown that longer stays in rehabilitation centers improve functional recovery [94, 95]. Ginis et al. review of literature recommended that patient should engage in 20 minutes of moderate to heavy aerobic activities with major muscle group strengthening exercises 2 times a week [96].
STEM CELL BASED TREATMENT FOR SCI
Stem cells have capabilities to self-renew and generate into more specialized cells. Therefore, they have been potentially useful for regenerative medicine [97, 98]. Furthermore, totipotent stem cells such as zygotes and some early stage cleavage-stage blastomeres have the potential to fully form an organism with a central nervous system (CNS) and peripheral nervous system (PNS) [99, 100]. Pluripotent stem cells such as embryonic stem cells (ESCs) have the potential to form all cells within the nervous system, but cannot form trophoblasts of the placenta [99].
The CNS has the potential to regenerate with NSCs. A NSC has been defined as a self-renewing cell with the ability to generate neural tissue [97, 99, 101]. Initially, NSCs were isolated through dissecting brain regions such as the subventricular zone (SVZ) and the hippocampus [98]. The tissue isolated was dissociated into component cells and exposed to growth factors such as fibroblast growth factor (FGF)-2 and epidermal growth factor (EGF). The differentiated cells were further isolated and selective cell lines were induced through withdrawing exposure to various factors [98]. Analysis was performed through staining with antibodies specific for astrocytes with glial fibrillary acidic protein (GFAP), oligodendrocytes with Ga1C and neurons with TUJ1 (neuronal beta III tubulin expressed in the CNS and PNS) [97].
Exogenous Transplantation Methods for SCI
Many types of stem cells have been considered for enhancing repair after SCI. Human ESCs were pluripotent, telomerase-positive and immortal cells that could self-renew and differentiate into multiple cell lineages [102-104]. In-vitro use of a 42 day differentiation protocol with H1 and H7 lines of human ESCs were shown to differentiate into oligodendrocyte progenitor cells (OPCs) [105, 106]. In rat models these OPCs enhanced axon remyelination and reestablished motor function within the injury site [107]. An important consideration in the clinical utility of human ESCs were the immunogenic consequences of allogenic cell transplantation [108]. OPCs were shown to be resistant to human natural killer (NK) cell destruction and the lytic effects of allogeneic and xenogeneic antibodies. Although human ESCs were associated with ethical dilemmas they have an advantage over bone marrow mesenchymal stem cells (BMMSCs). Conversely, the utilization of patient’s own BMMSCs was found to be beneficial for immune regulation, histocompatibility and repeated harvestability [109].
Stem Cells
Bone Marrow Stem Cells
Bone marrow stem cells (BMSCs) were shown to be relatively easy to isolate and grow in tissues cultures. They have been approved for use in hematopoietic diseases [110]. BMSCs consist of stromal cells and bone marrow (BM) containing nucleated cells [111-113]. Intravenous injection of both these types of cells demonstrated improved recovery of hind limb motor function after spinal cord compression in rats. Sykova et al. through their preclinical trials have demonstrated a benefit with different BMSC populations in both behavioral outcome and histopathological assessment [110].
Mesenchymal Stem Cells
Mesenchymal stromal cells were mesodermal, non-hematopoietic and easy to obtain. They were cultured ex-vivo and differentiated into neuronal lineages [114] (Refer to Fig. (2)). They were a target in acute, subacute and chronic SCI, and have been shown to migrate and integrate with injured spinal tissue, reduce cyst size and improve functional recovery [115].
Fig. (2)) Exogenous stem cell transplantation - Various sources of stem cell transplantation post spinal cord injury. Stem cells demonstrated in figure were neural stem/progenitor cells, (iPSCs), skin-derived precursors and MSCs. MSCs were obtained from the bone marrow, umbilical cord, adipose tissue, muscle and dental pulp from deciduous baby teeth. Dependent on culture conditions and growth factor exposure, differentiation of neural stem/progenitor cells can lead to OPCs, mature oligodendrocytes, astrocytes or neurons. Fibroblasts from the skin were reprogrammed into iPSCs and conversion of them directly to neurons and NSPCs without the need to pass into the pluripotent stage. J Clin Invest. 2012 Nov 1; 122(11):3824-3834; Copyright © 2012, American Society for Clinical InvestigationEmbryonic Stem Cells
Several groups have demonstrated in-situ oligodendrocyte differentiation and neurologic improvement after ESC implantation [116, 117]. Keirstead et al. through transplantation of human ESC-derived OPCs in rats continued to demonstrate oligodendrocyte differentiation, remyelination and locomotor improvement in acute injury within seven days [107]. Manley et al. supported the clinical use of OPC therapy for cervical SCI based on their preclinical efficacy and safety studies. This study used nude rats with cervical SCI lesions and was treated with AST-OPC1, a cell therapy product comprised of OPCs differentiated from the H7 human ESC line. After treatment with graft migration into the lesion site which suppressed the parenchymal cavitation there was improved motor behavioral recovery after four months. This was measured as locomotor performance. There was no effect on teratoma or tumor formation, development of allodynia or on mortality and morbidity rate [118].
Umbilical Cord Stem Cells
Liu et al. in six patients with complete SCI utilized umbilical cord mesenchymal stem cells (MSCs) demonstrated poor treatment response [119]. Tarasenko et al. demonstrated that primed (2×106 cells maintained for 7 days in Poly- D-lysine/Laminin-coated, 25-cm2 flasks of 4 ml of medium supplemented with 20 ng/ml basic FGF, 5 μg/ml heparin, and 0.5 μg/ml laminin) grafted human NSCs differentiated into cholinergic neurons in the intact spinal cord. When transplanted in mice 9 days after injury in the thoracic region the grafted NSCs survived for 3 months. They showed functional improvement of truncal stability. This was suggestive of functional improvement being dependent on timing of transplantation after injury, sites of grafting and sustainability of newly differentiated neurons and oligodendrocytes [120].
Adipose Derived Stem Cells
Forostyak et al. injected autologous adipose derived (AAD) MSCs intrathecally and found improvement in the American Spinal Injury Association (ASIA) motor and sensory scores after an 8 month follow up with nil magnetic resonance imaging interval changes [121].
Neural Stem Cells
NSCs have demonstrated to be multipotent cells with the ability to differentiate into neurons, oligodendrocytes and astrocytes. They were obtained from the developing and/or the adult CNS or were prepared as differentiated derivatives from the embryonic or induced pluripotent stem cells [122-128]. Since, there were ethical issues associated with obtaining fetal tissue, immortalized neuroepithelial stem cell lines have been developed. An example was cMycERTAM which was used to achieve conditional growth-promoting gene (c-myc) [129]. Induced pluripotent stem cells (iPSCs) were obtained from differentiated cells by reprogramming techniques through implanting specific transcription factors responsible for pluripotency [130] (Refer to Fig. (2)).
Many groups have demonstrated some degree of locomotor improvement after NSC grafting in SCI. Several studies demonstrated grafting of rat neural progenitor (NP) cells, human fetal spinal cord NP cells and human fetal NSCs after SCI [131-133]. Other studies have demonstrated transplantation of human brain-derived stem cells and human spinal cord tissue in injured mouse spinal cords [134-136]. Most grafted cells were shown to differentiate into glia. However, a small percentage differentiated into neurons [117, 131, 133]. Recovery of function noted in these studies were likely secondary to remyelination or release of trophic factors involved in survival and regeneration of host neurons from the grafted cells [69, 117, 133, 137-142].
Cummings et al. demonstrated that human CNS stem cells from fetal brain when grown as neurospheres were found to survive, migrate, engraft, differentiate and express markers for neurons and oligodendrocytes after SCI in NOD-scid mice. Conversely, the selective ablation of the grafted human neural cells by diphtheria toxin eliminated the observed benefits. Microscopically, new synapse formation between the grafted human NSCs and the host neurons was observed. Human NSCs when grafted after SCI in mice differentiated into gamma-aminobutyric acid (GABA) neurons [143].
Human NSCs derived from iPSCs have shown strong potential in experimental treatment of SCI [128, 144-146]. Ruzicka et al. found that use of iPSC-NP cells positively affected spinal cord regeneration by factors such glial scar formation, axonal sprouting, tissue sparing and cytokine levels. This lead to better outcomes in advanced locomotor tests (flat beam test and rotarod) [130].
Tarasenko et al. highlighted issues that needed to be addressed in NSC therapy in order to effectively replace lost neurons [120]. First issue was maintenance of these cells in culture. Second issue was methods to enhance neuronal differentiation of grafted stem cells in adults. Third issue was methods to directly stimulate stem cells to specific neuronal phenotypes. The intrinsic properties of grafted cells and microenvironment of host tissue were thought to guide phenotypic differentiation post grafting [147]. Wu et al. developed an in-vitro priming technology which lead to more than fifty percent of human fetal NSCs obtaining cholinergic phenotype in non-injured spinal cords [101]. Tarasenko et al. demonstrated that in-vitro priming of the grafted cells promoted differentiation to cholinergic phenotypic neurons in the post injury spinal cord [120]. Notably, the timing of grafting was important for graft survival [120, 148, 149]. Grafting of cells on day 9 was found to be most effective. Earlier grafting was less likely to survive due to detrimental effects of high levels of extracellular concentrations of glutamate and aspartate early after a SCI [150-152].
Other Cell Types for Transplantation
Schwann Cells Grafts
Schwann cells (SCs) present in grafts were involved in the growth promoting function when peripheral nerve grafts were transplanted into the CNS [153]. SCs were found to promote nerve fiber growth through secretion of neurotrophic factors, expressed cell adhesion molecules and various extracellular matrix molecules [154]. SCs not contacted by axons were found to release neurotrophic factors. SCs contacted by neurons stopped secreting the neurotrophic growth factors [155-158]. Xu et al. demonstrated that after placing SC grafts into 4-5mm spaces in the thoracic spinal cord of an adult rat that there was regeneration of more than 17,000 axonal processes. Barakat et al. found a three month survival of transplanted SCs in the chronic contusion model of SCI in rats [159]. There was regrowth of the corticospinal axons rostral to the injury graft interface in the descending serotonergic axons and the ascending calcitonin gene-related peptide positive fibers. There was reported modest recovery of locomotor function and other neurobiological indices [159, 160]. Martin et al. demonstrated that SC grafts were invaded by axonal profiles originating from the dorsal root ganglion neurons. There was an absence of descending fiber regeneration likely due to poor neurotrophic factor secretion and nil penetration of the supraspinal axons into the graft [161].
Olfactory Ensheathing Cells
Olfactory ensheathing cells (OECs) were not shown to be typical stem cells, but have demonstrated the ability to support neuronal regeneration in the olfactory and other areas of the CNS [162] (Refer to Fig. (3)). In SC transplantation, OECs were better integrated into the host glia, and had better migration along the white matter tracts [163]. OECs were present in the olfactory nerve layer, which was the outer layer of the olfactory bulb, and around the olfactory nerve layer [164-166]. Both areas contained two types of OECs, SC-like and astrocyte-like [167, 168]. SC-like OECs had diffuse glial-specific intermediate filament (IF) and GFAP staining with expression of low affinity growth factor (LAGF) p75 receptors [169, 170]. LAGF p75 receptors were downregulated after remyelination and regeneration of the axons with P0-positive (Schwann type) myelin. The astrocyte-like OECs had fibrous GFAP and expressed the embryonic form of neural cell adhesion molecule [168, 171, 172]. In-vitro, they have been found to transform from non-myelinating to myelinating [173]. Ramon-Cueto et al. demonstrated long-distance regeneration of spinal axons after transplanting OECs in a completely transected thoracic spinal cord [174]. Moreover, OECs have had a more directed elongation in regeneration of corticospinal axons along the lesion site, and into the distal part of the damaged white matter tract [175]. OECs were also shown to remyelinate axons, which have been chemotoxically demyelinated [176]. In sum, OECs were found to establish a glial environment, promote axonal growth, increase angiogenesis, prevent cavity formation and modify scar formation [177].
Fig. (3)) Olfactory ensheathing cell transplants - Promoting of anatomical and synaptic plasticity through olfactory ensheathing cell transplants. This figure demonstrated that both anatomical sprouting and synaptic plasticity could improve function within the zone of partial preservation above a complete/partial injury and would maximize the function of spared fibers passing the lesion site.(1) Shows release of trophic factors by olfactory ensheathing cell (OEC) transplants promoting sprouting of intact fiber systems.(2) Shows release of neurotrophic factors released from OEC transplants and induce an increase in the efficacy of synaptic transmission of the surviving connections. The inner square (in yellow) represents the area of injury. Nature Reviews Neurology. 2007 Mar 1; 3:152-161; Copyright © 2007, Springer Nature.Endogenous Stem Cells for SCI
Neuronal regeneration has become an area of research with potential for clinical application. Although primarily at the animal stage, previous and current studies as discussed below have shown to be promising for eNSCs.
Altman et al. introduced the notion of new neurons in the adult rodent brain [178]. SCI research initially emphasized regrowth and reconnection techniques of severed corticospinal tracts through re-innervating alpha motor neurons in the ventral horns [179]. More recently, there has been an up rise in attempts to use nascent local spinal interneurons to form a neuronal relay network. This would serve as a “bridge” to circumvent long-distance axonal growth [180, 181]. Adult neurogenesis was found to be an active process of proliferation, fate specification of NPs, differentiation, maturation, navigation and functional integration of neurons into the existing neuronal circuitry [182]. It was also found to be a dynamic process regulated by stimuli and modulated by the existing neuronal circuitry [183]. Neurogenesis occured in the normal brain in new olfactory interneurons and hippocampal granule cells [184]. 5-ethynyl-2'-deoxyuridine labelling of mice brains has shown that the main proliferative zone has proliferative potential. However, all proliferative cells were not likely to be differentiated into neurons and incorporated into neuronal circuitry [185]. Compared to postnatal development, adult neurogenesis had a more prolonged process. Adult borne dentate granule cell development, formation and maturation lasted more than eight weeks. Postnatally, a similar process occurred within two weeks [186, 187].
Advantages of Endogenous versus Exogenous Stem cells
The utilization of eNSCs has become appealing as they are noninvasive, avoid need for immunosuppression and concern for immune rejection [188]. Moreover, they avoid associated ethical dilemmas and concern for tumor formation secondary to dysregulated cell proliferation [189].
Areas of Neurogenesis in Spinal Cord
eNSCs have shown to exist in the brain and the spinal cord. The adult mammalian spinal cord was a non-neurogenic region. In non-pathological situations the progenitor cells were glial-restricted leading to oligodendrocytes and astrocytes formation, but not neurons. NSCs were located in the white matter parenchyma and close to the ependymal/subependymal layer (SEL) in the central canal [190-195]. Two models exist on the location of neural stems cells in the intact spinal cord [190]. The first model postulated that the multipotent stem cells resided in the ependymal layer of central canal, known as ependymal stem/progenitor cells (epSPCs). Here, they divided asymmetrically into daughter cells and NP cells [191, 193]. They further migrated to the outer portion of the spinal cord becoming either proliferative glial progenitor (GP) cells or mature glial cells of the spinal cord [196]. The second model postulated that the stem cells and GP resided independent of the proliferating ependymal cells (ECs) in the spinal cord parenchyma [196, 197]. NP cells in the spinal cord white matter expressed markers such as chondroitin sulphate proteoglycan neuron-glial antigen (NG) 2 and transcription factors such as oligodendrocyte transcription factor (Olig2) and NKx2.2. This represented a spectrum ranging from multipotent to restricted GP cells designated to the oligodendrocyte lineage [190, 194, 198].
Histology of the Central Spinal Cord
The ependymal and periependymal region of the central spinal cord region consisted of tanycytes, ependymocytes and cerebrospinal fluid (CSF)-contacting neurons [199]. Tanycytes, involved in communication between CSF and capillaries radiated from the central canal into the gray matter and terminated on blood vessels [199, 200]. Ependymocytes were ciliated cells and connected with each other through gap junctions [201]. CSF-contacting neurons were found in lower vertebrates. They have been shown to have similar electrophysiological properties similar to immature neurons, and communicate with local neurons at higher spinal segments. They were responsible in maintaining a potential of hydrogen in the CSF through P2X receptors, and express neuronal nuclei (NeuN) and doublecortin neuronal lineage markers [202-204]. Studies have demonstrated that cells in the SEL had increased proliferative capacity after a SCI event [205, 206]. Post injury, cells in mice expressed NSC markers, migrated to the site of injury in the spinal cord and differentiated into astrocytes and oligodendrocytes. Therefore they were key components in forming the glial scar and forming the functional neuronal circuits [193, 205-209] (Refer to Fig. (4)).
Forms of Stem Cells in the Spinal Cord
OPCs, ECs and astrocytes were postulated as potential stem cells in the spinal cord [190-194]. Studies by several groups indirectly found that OPCs had NSC properties, and formed multiple lineages in the spinal cord [194, 198, 210, 211]. Others found that ECs consisted of the main cell population in the intact spinal cord, and gave rise to differentiated progeny after injury. Although ECs had in-vitro NSC properties, their origin after injury was not known [193, 212]. Buffo et al. found that astrocytes had minimal proliferative activity with only in-vitro NSC properties after injury in the cortical astrocytes [213]. They only had stem cell properties in the SVZ and dentate gyrus [214]. Astroglial activation included two sources: reactivated resting astrocytes and new astrocytes from eNSCs [116].
Shihabuddin et al. showed that transplantation of NSCs obtained from the spinal cord into the hippocampal dentate gyrus gave rise to neurons. Furthermore, with re-implantation of NSCs in the spinal cord they continued to differentiate into glial cells. This study had two implications. First, the NSCs isolated from the spinal cord were multipotent. Second, the NSCs obtained from the spinal cord were not fate restricted, but restricted to express their multipotency in the spinal cord. This was likely due to environmental and intrinsic factors regulating proliferation, migration and differentiation in the spinal cord [215].
Fig. (4)) Glial scar formation - Cellular composition post spinal cord injury. Figure demonstrated the heterogeneous neural cell infiltrate in the glial scar post spinal cord injury.(Top left) showed location of neural cells in the intact spinal cord(Bottom right) showed pathology of spinal cord injury with layers of the healing process and differential of the neural cells post injury. Neurosci Bull. 2013 Jan 1; 29(4):421-435; Copyright © 2013, Springer NatureEnvironmental Support for Stem Cells
NSC environments were found to be nurtured with local astrocytes [216-219]. They were present throughout the total life span of the organism [220]. Astroglial cell staging was important in supporting or inhibiting NSC migration [221]. Primary astrocytes from neurogenic locations have the ability to promote neurogenic differentiation [222]. Non-neurogenic areas in mice could be induced with injecting subgranular zone astrocytes and sonic hedgehog [217]. Astrocytes were not fate determined, and under specific conditions differentiated accordingly [223-228].
IF has been shown to have both structural and non-structural function in eukaryotes [229]. The non-structural function of IF was an important component of signaling pathways for cell survival and migration [230]. Nestin was transiently expressed in NSCs, and down regulation of its expression was related to differentiation of cells into astrocytes, neurons and oligodendrocytes. It has been found that nestin expression of active astrocytes in mice was mediated through EGF-ErbB1. Further intracellular interactions were through the Ras, Raf, MEK and extracellular signal-regulated kinase (ERK) pathways [220, 223, 231]. Nestin was not expressed in mature cells [220]. However, during acute injury, nestin expression was suggestive of active proliferation of progenitor astrocytes [223]. Same gene sequences controlling nestin during CNS development were involved in up-regulating nestin in astrocytes during states of CNS injury [220, 231, 232]. EGF through the EGF receptor - ErbB1 was involved in transformation into active astrocytes [233].
Yang Z et al. demonstrated the importance of establishing a nurturing microenvironment for eNSCs and ExSCs to stimulate nascent neural circuitry in the neural repair process [179]. Through a biodegradable material chitosan, a slow release of neurotrophin-3 was achieved in the microenvironment over 14 weeks. This allowed eNSCs to be activated, and later differentiate. The neutrotrophin-3- chitosan model functions through the neutrophin-3 (NT3)-tropomyosin receptor kinase (Trk) C signaling. The three modes of actions in new neuronal growth were: long distance nerve fiber growth through the formation of a nascent neural synaptic network, inhibition of inflammatory immune activity and stimulation in formation of blood vessels [179, 234, 235].
Implications of Spinal Cord Injury and its Effects on Regeneration
Hofstetter et al. based on better functional recovery after in-vitro transplantation of NSCs found that the adult spinal cord had stem cells which were activated by injury, but were unable to exert the beneficial effects in normal conditions [236]. SCI activated astrocytes, fibroblasts and macrophages which were involved in forming a scar to hinder further injury [237]. This scar was proposed to be detrimental because it hindered axonal regeneration and acted as a physical barrier [238-240]. The molecular and cellular mechanism of scar formation was altered at the messenger ribonucleic acid (RNA) level [192]. After a SCI, MicroRNA-486 was responsible for regulation of inflammation. miR-20a was involved in cell death and miR-21 in astrocyte activity. Although they were “actively” involved in these processes. There was no evidence that they directly acted on NSCs [241-243]. Moreover, long non-coding RNAs have also been found to affect posttranscriptional regulation of SCI events [244-246]. Wang et al. demonstrated that knockdown of IncSCIR1 caused astrocyte proliferation and migration. There was a strong correlation between the downregulation of IncSCIR1, decrease of Wnt3 and increase of bone morphogenetic protein (BMP) 7. Therefore, local overexpression of IncSCIR1 promoted functional recovery with an inhibitory effect on the environment around the location of injury [247].
Glial Scar Debate
Moore et al. examined the histochemical changes after a SCI [248]. They found that there was a 3-fold increase in GFAP expression in the SEL at the site of lesion and at proximal sites in the canine spinal cord. The basis of these findings was multifold. First, there was increased astrocytic differentiation of neural precursor cells (NPCs) in SEL. Second, astrocytes were derived from the EC layer and migrated to the injury site with regulatory effects of the inflammation. Third, the glial scar formation process involved astrocyte migration into the injured region [209, 248-250] (Refer to Fig. (4)). Moore et al. findings suggested that this process was diffuse and occurred throughout the spinal cord. NSCs transforming into astroglia was possibly a natural protective mechanism to confine injury size, but later contributed to the glial scar and hindrance in regrowth of axons. In addition, neurons and oligodendrocytes were the main cells dying after SCI. NSCs were the potential source to replace them. This enhanced neuronal and oligodendrocyte (ODC) differentiation towards eNSC-based recovery [248].
Anderson et al. assessed the role of reactive astrocytes in axon regeneration in post-experimental SCI mouse models. They prevented the formation of reactive astrocytes through selectively killing proliferating scar-forming astrocytes, deleting STAT3 transcription factor, genetically altering the astrocytes to express a diphtheria toxin and killing astrocytes through ultralow doses of diphtheria toxin [251]. In animals, lacking the glial scar yielded poor spontaneous regrowth of damaged axons and increased degeneration of axons from the distal to the proximal end of injury. When compared with controls, areas with nil astrocyte scar formation did not contain any axons. This suggested the importance of chronic astrocyte scar formation in preserving the integrity of the neuronal tissue. They also demonstrated that chondroitin sulfate proteoglycans were significantly higher in the injured spinal cord compared to not injured controls. Further removal of the scar did not decrease the chondroitin sulfate proteoglycan levels. Aggrecan, a growth inhibiting chondroitin sulfate proteoglycan was not detected in scar forming astrocytes. It was also demonstrated that injecting neurotrophins and brain-derived neurotrophic factor (BDNF) into the injury site stimulated axonal growth through the astrocytic scar. Removing the scar inhibited axonal regeneration [251].
Hub Gene Network Analysis
Duan et al. used the Weighted Gene Coexpression Analysis to establish hub network genes which regulate microenvironment functions. Hub gene network analysis demonstrated modules (Cm) that were positively and negatively correlated with NT3-chitosan treatment. Cm1 and 2 were involved in neurogenesis. Cm3 was involved in pain and temperature sensory processing. Cm4 was involved in muscle contraction. Cm5 was involved in angiogenesis. Cm6 was involved in immune response. Cm7 was involved in the wound, stress and inflammatory response. Cm1,2,3,4 and 5 were positively correlated with NT3-chitosan. Cm6 and 7 were negatively correlated with NT3-chitosan [235]. Yang et al. showed that corticospinal axons made contact with new neurons. This was through labelling the corticospinal motor neurons with biotin, fluorescein and dextran to Bromodeoxyuridine (BrdU)/microtubule associated protein 2 with the new neurons in the neurotrophin-3-chitosan model. To assess for functional recovery, cytosine beta-D-arabinofuranoside was used to decrease nestin-positive NSCs and new NeuN-positive neurons. The treatment group showed 30-40% decrease in Basso Beattie Bresnahan scores in the bilateral hind limbs at 1 year. Moreover a decrease in amplitude by 30% with nil decrease in latency was noted in somatosensory evoked potentials (SEP) and motor evoked potentials (MEP) after treatment. This suggested that cytosine beta-D-arabinofuranoside did not alter the number of regenerating axons. Change in amplitudes suggested that locomotor functional recovery was due to regeneration of neurons [179].
Purinergic Receptors
Gómez-Villafuertes et al. demonstrated the properties of the purinergic receptors altering the intrinsic properties of the epSPCs by enhancing regulation of proliferation, differentiation and lineage specification after a SCI [252, 253]. Nucleotides activated two different types of purinergic receptors called P2X and P2Y. P2X (P2X 1-7) were ion-gated channels involved in fast calcium influx (ionotropic). P2Y (P2Y 1-14) were G-protein-coupled receptors (metabotropic). NP cells found in adult SVZ and subgranular layer of the hippocampal dentate gyrus of the hippocampus had functional purinergic receptors [254]. Moreover, neurospheres obtained from fetal rat brain expressed P2X2-P2X7 and P2Y1,2,4,6. Adult mouse SVZ expressed P2Y1 and P2Y2 purinergic receptors types [255-258].
After a SCI, there were two effects of activation of the purinergic receptors in the area of lesion. First, there was an activation of purinergic receptors due to release of large amounts of adenosine triphosphate (ATP) and nucleotides by the injured tissues. Due to the interaction of various growth factors there was remodeling and repair of the lesion [255, 259]. Studies have shown that the NP cells have been an important source of local ATP. This was supported by purinergic receptors being localized in regions of mitotic progenitor cell expansion and neurogenesis in the adult brain [254, 260, 261]. External ATP supplementation increased the mitotic index and rate of NP cells [260]. Second, purinergic receptor interaction led to decreased gliosis and glial scar formation [262].
Gómez-Villafuertes et al. through microfluorimetric techniques using calcium dye Fura-2 demonstrated that nucleotide activation through ionotropic P2X4,7 and metabotropic P2Y1,4 led to epSPC response. P2X7 receptor was shown to have dual functions. First, it had an inhibitory effect on neurogenesis and axon out-growth. Second, it was a cell death inducer [252]. Other studies have found that P2X7 receptor inhibition have led to cultured hippocampal neuron axonal growth and branching [263]. Activation of P2X7 receptors has led to necrosis and apoptosis of embryonic and adult NPCs. This could adversely affect NP cell survival after a SCI secondary to excessive neuro or gliogenesis [264, 265]. P2Y1,2 have been found to stimulate NP cell migration which facilitates movement of NSCs in the neurogenic niches [257, 266]. Moreover, it could have played a crucial role in NP cell expansion. Further, which could have helped maintain NSC/NP cell niches in adult brains [252]. P2Y1 receptor antagonists aided NSC differentiation into neurons and glial cells through decreasing the size and frequency of primary neurospheres [267]. epSPCs were found to express functional P2Y1 and P2Y4 receptors [252]. P2Y receptor antagonists were found to suppress mitotic index of cells, which suppressed neurosphere expansion [260]. Severe spinal cord contusions have led to early and sustained increase in P2X4,7 receptors [252]. de Rivero Vaccari JP et al. found that acute transplantation of undifferentiated epSPCs which were derived from rats with SCI reversed the effect [265]. P2X4 knockout mice demonstrated decreased levels of neuroinflammation after SCI leading to improved functional recovery during the first week after injury [268]. Use of P2X7 receptor was controversial after a SCI as some studies demonstrated failed motor and histopathological improvement. Other studies have shown that P2X7 antagonism improved function and reduced the pathological consequences of SCI [252, 269].
Genetic Fate Mapping
Although the findings of adult neurogenesis and presence of NSCs have been encouraging. However, there has been difficulty in determining the identity and in localizing the cells in the spinal cord [190, 270]. Therefore, the utility of genetic fate mapping (GFM) has been used to characterize, identify and aid in localizing potential NSC populations [123]. The principle of GFM involves labeling of precursor cells, and then tracing their lineage both in-vivo and in-vitro. It was also a technique used in determining cell behaviors such as proliferation, movement and lineage exclusion [270, 271].
Types of Labelling Techniques
Different types of labelling techniques were used to delineate different cell lineages. Fluorescent labeling of neurosphere-initiating cells demonstrated that multipotent NSCs resided close to the central canal of the spinal cord [191, 192]. Several groups showed that NSCs were stimulated from the medial and lateral parts of the spinal cord and were able to expand passages up to two times [195, 272]. Indirect methods to label cells decreased specificity of detection of NSCs [273]. BrdU labeling involved BrdU being incorporated and substituting thymidine into the newly synthesized DNA during the S phase of the cell cycle. BrdU specific antibodies were used to detect the incorporated chemical which was suggestive of active replication of DNA [274].
Labelling with BrdU was achieved through drinking water for 5 weeks which predominantly marked approximately 80% of ECs, astrocytes and oligodendrocytes. Minority cells labelled were microglia and blood vessel associated cells in the spinal cord [116, 190]. These labelled cells proliferated in normal conditions and after injury, but did not provide lineage data [190, 198, 275]. Retroviral infections were limited in their mapping function as they labelled only a small population of dividing cells with low specificity [194, 198].
