Exogenous leukemia inhibitory factor stimulates oligodendrocyte progenitor cell proliferation and enhances hippocampal remyelination

New CNS neurons and glia are generated throughout adulthood from endogenous neural stem and progenitor cells. These progenitors can respond to injury, but their ability to proliferate, migrate, differentiate, and survive is usually insufficient to replace lost cells and restore normal function. Potentiating the progenitor response with exogenous factors is an attractive strategy for the treatment of nervous system injuries and neurodegenerative and demyelinating disorders. Previously, we reported that delivery of leukemia inhibitory factor (LIF) to the CNS stimulates the self-renewal of neural stem cells and the proliferation of parenchymal glial progenitors. Here we identify these parenchymal glia as oligodendrocyte (OL) progenitor cells (OPCs) and show that LIF delivery stimulates their proliferation through the activation of gp130 receptor signaling within these cells. Importantly, this effect of LIF on OPC proliferation can be harnessed to enhance the generation of OLs that express myelin proteins and reform nodes of Ranvier in the context of chronic demyelination in the adult mouse hippocampus. Our findings, considered together with the known beneficial effects of LIF on OL and neuron survival, suggest that LIF has both reparative and protective activities that make it a promising potential therapy for CNS demyelinating disorders and injuries.     

The potential of neural stem cells to repair stroke-induced brain damage

Acute injuries to CNS such as stroke induce neural progenitor proliferation in adult brain which might be an endogenous attempt to self-repair. This process is known to be altered by several exogenous and endogenous modulators including growth factors that could help to reinforce the post-stroke neurogenesis. Increasing the neurogenesis may be a future therapeutic option to decrease the cognitive and behavioral deficits following stroke. In addition, transplantation of various types of stem cells into the injured brain is currently thought to be an exciting option to replace the neurons lost in the post-ischemic brain. These include immortalized stem cell lines, neural progenitors prepared from embryonic and adult animals and mesenchymal stem cells. Using exogenous stem cells in addition to modulating endogenous neurogenesis, we may be able to repair the injured brain after a devastating stroke. This article reviewed the current literature of these two issues.     

Improved neural progenitor cell survival when cografted with chromaffin cells in the rat striatum

Transplantation of stem and neural progenitor cells hold great promise in the repair of neuronal tissue lost due to injury or disease. However, survival following transplantation to the adult CNS has been poor, likely due to a lack of neurotrophic factors, such as basic fibroblast growth factor (FGF-2), that are used to maintain and expand these cells in culture. Chromaffin cells produce several neurotrophic agents, including FGF-2, which may aid in both neuroprotection following injury and progenitor cell proliferation and survival. In addition, increased CNS catecholamines have been shown to improve functional recovery following insult. Thus, cotransplants of neural progenitor cells and chromaffin cells may be a useful clinical strategy. To address this, the survival of rat cortical progenitors transplanted to the adult rat striatum with and without bovine chromaffin cell cografts was assessed. Progenitors obtained from E14 embryos were prelabeled with bromodeoxyuridine (BrdU) before transplantation to enable later identification. Transplants were made both unilaterally and bilaterally, where animals received a monograft (progenitor cells alone) on one side and a cograft (progenitors + chromaffin cells) on the other. Histological results after 7, 17, and 30 days posttransplant revealed greatly improved survival of BrdU-labeled cells in the cografts and also less infiltration of presumptive immune cells. In addition, perivascular cuffing was seen in the monografts. In vitro progenitor cohorts stained positive for nestin, GFAP, and beta-tubulin III, but in vivo very few cells were found that were double labeled with BrdU and one of these markers. Thus, in contrast to in vitro findings, chromaffin cells did not enhance differentiation of progenitors in vivo during the 30 days posttransplantation. The results of these studies suggest that chromaffin cells may provide neurotrophic support to enhance survival, but not differentiation, of cortical progenitor grafts in the adult CNS.     

Enhanced viability and neuronal differentiation of neural progenitors by chromaffin cell co-culture

The transplantation of neural stem cells and progenitors has potential in restoring lost cellular populations following central nervous system (CNS) injury or disease, but survival and neuronal differentiation in the adult CNS may be insufficient in the absence of exogenous trophic support. Adrenal medullary chromaffin cells produce a trophic cocktail including basic fibroblast growth factor (FGF-2) and neurotrophins. The aim of this study was to evaluate whether chromaffin cells can provide a supportive microenvironment for neural progenitor cells. In order to assess this, the growth and differentiation of neural progenitor cell cultures from embryonic rat cortex were compared in standard FGF-2-supplemented neural progenitor growth media, in standard media but lacking FGF-2, or in media lacking FGF-2 but co-cultured with bovine chromaffin cells. Using bromodeoxyuridine (BrdU)-prelabeling, findings indicated poor survival of progenitor cultures in the absence of FGF-2. In contrast, the addition of chromaffin cells in co-culture appeared to 'rescue' the progenitor cultures and resulted in robust neurospheres containing numerous BrdU-labeled cells interspersed with and closely apposed to chromaffin cells. As indicated by H3 labeling, cells in co-cultures continued to proliferate, but at a substantially reduced rate compared with standard FGF-2 supplemented growth media. The co-cultures contained more beta-tubulin III-positive processes than parallel cultures maintained in FGF-2-supplemented media and these cells displayed a more mature phenotype with numerous varicosities and complex processes. These findings indicate that chromaffin cells can provide a supportive environment for the survival and neuronal differentiation of neural progenitor cells and suggest that their addition may be useful as a sustained source of trophic support to improve outcomes of neural stem cell transplantation.     

Fate of endogenous stem/progenitor cells following spinal cord injury

The adult mammalian spinal cord contains neural stem and/or progenitor cells that slowly multiply throughout life and differentiate exclusively into glia. The contribution of adult progenitors to repair has been highlighted in recent studies, demonstrating extensive cell proliferation and gliogenesis following central nervous system (CNS) trauma. The present experiments aimed to determine the relative roles of endogenously dividing progenitor cells versus quiescent progenitor cells in posttraumatic gliogenesis. Using the mitotic indicator bromodeoxyuridine (BrdU) and a retroviral vector, we found that, in the adult female Fisher 344 rat, endogenously dividing neural progenitors are acutely vulnerable in response to T8 dorsal hemisection spinal cord injury. We then studied the population of cells that divide postinjury in the injury epicenter by delivering BrdU or retrovirus at 24 hours after spinal cord injury. Animals were euthanized at five timepoints postinjury, ranging from 6 hours to 9 weeks after BrdU delivery. At all timepoints, we observed extensive proliferation of ependymal and periependymal cells that immunohistochemically resembled stem/progenitor cells. BrdU+ incorporation was noted to be prominent in NG2-immunoreactive progenitors that matured into oligodendrocytes, and in a transient population of microglia. Using a green fluorescence protein (GFP) hematopoietic chimeric mouse, we determined that 90% of the dividing cells in this early proliferation event originate from the spinal cord, whereas only 10% originate from the bone marrow. Our results suggest that dividing, NG2-expressing progenitor cells are vulnerable to injury, but a separate, immature population of neural stem and/or progenitor cells is activated by injury and rapidly divides to replace this vulnerable population.     

Molecular regulation of adult CNS neurogenesis: an integrated view

Neural stem cells in the adult subventricular zone and dentate gyrus might be utilized to replace lost neurons in neurological disorders. The development of treatments would be facilitated by identifying the mechanisms that contribute to functional neurogenesis in adult animals. This review focuses on the emerging view that localized and overlapping pathways of growth factors, metalloproteases, neurotransmitters and hormones regulate different aspects of neurogenesis within the neurogenic niches. Neuroblast migration is precisely regulated by cooperation between several repellants, attractants and guidance molecules that are located within specific CNS regions. Further elucidation of crucial molecular regulators and integration of their signaling cascades should lead to more rational and effective approaches to harness the exciting phenomenon of adult CNS neurogenesis.     

Stem and progenitor cell therapies: recent progress for spinal cord injury repair

Mechanical trauma to the spinal cord is often accompanied by irreversible tissue damage, limited endogenous repair and permanent loss of motor, sensory and autonomic function. The implantation of exogenous cells or the stimulation of endogenous cells, to repopulate and replace or to provide a conducive environment for repair, offers a promising therapeutic direction for overcoming the multitude of obstacles facing successful recovery from spinal cord injury. Although relatively new to the scene of cell based therapies for reparative medicine, stem cells and their progenitors have been labeled as the 'cell of the future' for revolutionizing the treatment of CNS injury and neurodegenerative disorders. The following review examines the different types of stem cells and their progenitors, their utility in experimental models of spinal cord injury and explores the outstanding issues that still need to be addressed before they move towards clinical implementation.     

Induced pluripotent stem cell-derived neural stem cell therapies for spinal cord injury

The greatest challenge to successful treatment of spinal cord injury is the limited regenerative capacity of the central nervous system and its inability to replace lost neurons and severed axons following injury. Neural stem cell grafts derived from fetal central nervous system tissue or embryonic stem cells have shown therapeutic promise by differentiation into neurons and glia that have the potential to form functional neuronal relays across injured spinal cord segments. However, implementation of fetal-derived or embryonic stem cell-derived neural stem cell therapies for patients with spinal cord injury raises ethical concerns. Induced pluripotent stem cells can be generated from adult somatic cells and differentiated into neural stem cells suitable for therapeutic use, thereby providing an ethical source of implantable cells that can be made in an autologous fashion to avoid problems of immune rejection. This review discusses the therapeutic potential of human induced pluripotent stem cell-derived neural stem cell transplantation for treatment of spinal cord injury, as well as addressing potential mechanisms, future perspectives and challenges.     

Stem cell repair of central nervous system injury

Neural stem cells (NSCs) have great potential as a therapeutic tool for the repair of a number of CNS disorders. NSCs can either be isolated from embryonic and adult brain tissue or be induced from both mouse and human ES cells. These cells proliferate in vitro through many passages without losing their multipotentiality. Following engraftment into the adult CNS, NSCs differentiate mainly into glia, except in neurogenic areas. After engraftment into the injured and diseased CNS, their differentiation is further retarded. In vitro manipulation of NSC fate prior to transplantation and/or modification of the host environment may be necessary to control the terminal lineage of the transplanted cells to obtain functionally significant numbers of neurons. NSCs and a few types of glial precursors have shown the capability to differentiate into oligodendrocytes and to remyeliate the demyelinated axons in the CNS, but the functional extent of remyelination achieved by these transplants is limited. Manipulation of endogenous neural precursors may be an alternative therapy or a complimentary therapy to stem cell transplantation for neurodegenerative disease and CNS injury. However, this at present is challenging and so far has been unsuccessful. Understanding mechanisms of NSC differentiation in the context of the injured CNS will be critical to achieving these therapeutic strategies.     

Adult neurogenesis pharmacology in neurological diseases and disorders

With the confirmation that neurogenesis occurs in the adult brain and neural stem cells reside in the adult CNS, the focus of research has now shifted to the understanding of the function of newborn neuronal cells in the adult brain, and particularly in the pathologies of the nervous system. Neurogenesis has been reported to be modulated in a broad range of pathological conditions, including neurological diseases and disorders. More strikingly, studies have revealed that drugs currently used to treat neurological diseases and disorders, such as Alzheimer's disease and depression, increase adult neurogenesis, which may mediate their activities. However, some of these studies are the source of debates and controversies, and remain to be confirmed. Hence, the role and contribution of newly generated neuronal cells in neurological diseases and disorders, as well as the effect of drugs on adult neurogenesis and its significance remain to be elucidated and understood. This shows that adult neurogenesis is not only important for our understanding of development and therapy, but also for the physiopathology of the CNS and its pharmacology.     

Neuronal replacement in the injured olfactory bulb

The adult forebrain subventricular zone contains neural stem cells that produce neurons destined for the olfactory bulb, where interneuron populations turnover throughout life. Forebrain injuries can stimulate production of these cells, and re-direct migrating precursors from the olfactory system to areas of damage, where their region-appropriate differentiation and long-term functional integration remain a matter for debate. Paradoxically, little is known about the ability of these progenitors to replace olfactory neurons lost to injury. Their innate capacity to generate bulb neurons may give them an advantage in this regard, and using injections of N-methyl-d-aspartate to kill mature olfactory bulb neurons, combined with bromodeoxyuridine labeling to monitor the fate of adult-born cells, we investigated the potential for injury-induced neurogenesis in this system. Widespread degeneration of bulb neurons did not affect the rate of cell proliferation in the subventricular zone, or cause neuroblasts to divert from their normal migratory route. However migration was slowed by the injury, leading to the accumulation and differentiation of neuroblasts as NeuN+ cells in the rostral migratory stream within 2 weeks of their birth. Despite this, a subset of new neurons successfully invaded the damaged bulb tissue, where they expressed neuronal markers including NeuN, calretinin, GABA, and tyrosine hydroxylase, with some surviving here for as long as 6 months. To test for functional integration of cells born post-injury, we also performed smaller NMDA lesions in restricted portions of the bulb granule cell layer and observed adult-born NeuN+ cells in these areas within 5 weeks, and BrdU+ cells that expressed the immediate-early gene c-fos following odor stimulation. These data suggest that the normal neurogenic capacity of the adult subventricular zone can be adapted to replace subsets of olfactory neurons lost to injury.     

Genetically engineered human neural stem cells for brain repair in neurological diseases

Neural stem cells (NSCs)of the central nervous system (CNS) have recently received a great deal of attention and interest for their therapeutic potential for neurological disorders. NSCs are defined as CNS progenitor cells that have the capacity for self-renewal and multipotent potential to become neurons or glial cells. Recent studies have shown that NSCs isolated from mammalian CNS including human can be propagated in vitro and then implanted into the brain of animal models of human neurological disorders. Recently, we have generated clonally derived immortalized human NSC cell lines via a retroviral vector encoded with v-myc oncogene. One of the human NSC lines, HB1.F3, was utilized in stem-cell based therapy in animal models of human neurological disorders. When F3 human NSCs were implanted into the brain of murine models of lysosomal storage diseases, stroke, Parkinson disease, Huntington disease or stroke, implanted F3 NSCs were found to migrate to the lesion sites, differentiate into neurons and glial cells, and restore functional deficits found in these neurological disorders. In animal models of brain tumors, F3 NSCs could deliver a bioactive therapeutically relevant molecules to effect a significant anti-tumor response intracranial tumor mass. Since these genetically engineered human NSCs are immortalized and continuously multiplying, there would be limitless supply of human neurons for treatment for patients suffering from neurological disorders including stroke, Parkinson disease, Huntington disease, ALS, multiple sclerosis and spinal cord injury. The promising field of stem cell research as it applies to regenerative medicine is still in infancy, but its potential appears limitless, and we are blessed to be involved in this exciting realm of research.     

Region-specific differentiation of embryonic stem cell-derived neural progenitor transplants into the adult mouse hippocampus following seizures

Embryonic stem (ES) cells can generate neural progenitors and neurons in vitro and incorporate into the adult central nervous system (CNS) following transplantation, suggesting their therapeutic potential for treating neurological disorders. However, our understanding of the conditions that direct ES-derived neural progenitor (ESNP) migration and differentiation within different regions of the adult CNS is incomplete. Rodents treated with the chemoconvulsant kainic acid (KA) experience seizures and display hippocampal sclerosis, as well as enhanced hippocampal neurogenesis, similar to pathological findings in patients with temporal lobe epilepsy (TLE). To examine the potential for ESNPs to incorporate into the adult hippocampus and differentiate into hippocampal neurons or glia following seizure-induced damage, we compared the fates of ESNPs after they were transplanted into the CA3 region or fimbria 1 week following KA-induced seizures. After 4-8 weeks, ESNPs grafted into the CA3 region had migrated to the dentate gyrus (DG), where a small subset adopted neural stem cell fates and continued to proliferate, based on bromodeoxyuridine uptake. Others differentiated into neuroblasts or dentate granule neurons. In contrast, most ESNPs transplanted into the fimbria migrated extensively along existing fiber tracts and differentiated into oligodendrocytes or astrocytes. Hippocampal grafts in mice not subjected to seizures displayed a marked tendency to form tumors, and this effect was more pronounced in the DG than in the fimbria. Taken together, these data suggest that seizures induce molecular changes in the CA3 region and DG that promote region-specific neural differentiation and suppress tumor formation.     

Adult neurogenesis and neuroplasticity

After cerebral strokes and traumatic brain injuries (TBIs), there is a striking amount of neurological recovery in the following months and years, despite often-permanent structural damage. Though the mechanisms underlying such recovery are not fully understood, properties of plasticity of the central nervous system (CNS), such as the reorganization of the pre-existing network and axonal sprouting have been implicated in the recovery. With the recent evidences that neurogenesis occurs in the adult brain, and neural stem cells (NSCs) reside in the adult CNS, the involvement of newly generated neuronal cells in the recovery following injury to the CNS remains to be established. Neurogenesis is increased bilaterally in the dentate gyrus (DG) and the subventicular zone (SVZ) after cerebral strokes and TBIs, and new neuronal cells are generated at the sites of injury, where they replace some of the degenerated nerve cells. Newly generated neuronal cells at the sites of injury may represent an attempt by the CNS to regenerate itself after injury, whereas the increased neurogenesis in the DG and SVZ would also contribute to the CNS plasticity. Thus, injury-induced neurogenesis may contribute to the recovery and plasticity of the CNS.     

Adult stem cell therapy in stroke

PURPOSE OF REVIEW: Acute cerebral infarction causes irreversible locally restricted loss of the neuronal circuitry and supporting glial cells with consecutive functional deficits and disabilities. The currently available and effective therapy targets fast vessel recanalization accompanied by symptomatic measures. Research activities focusing on stem cells, which represent a promising source for organotypic cell replacement and functional recovery after stroke, have gained momentum in recent years, making regenerative cell-based therapies a much more feasible realistic approach. This review provides an update about preclinical and clinical cell-based studies in stroke focusing on stem cells derived from the adult central nervous and hematopoetic systems. RECENT FINDINGS: Endogenous neural stem cells, which have been shown to reside throughout life in the central nervous system, have the capacity to replace lost neurons in models for numerous disorders, including cerebral ischemia. Considering adult neural stem cell transplantation as a regenerative strategy after stroke, progress has been made in isolating human adult neural stem cells and demonstrating the feasibility of autologous neural stem cell transplantation. An increasing number of studies provide evidence that hematopoietic stem cells, either after stimulation of endogenous stem cell pools or after exogenous hematopoietic stem cell application (transplantation), improve functional outcome after ischemic brain lesions. Various underlying mechanisms such as transdifferentiation into neural lineages, neuroprotection through trophic support, and cell fusion have been deciphered. SUMMARY: Many preclinical studies employing adult stem cell-based strategies hold great promise. For endogenous approaches the correlate of cell replacement underlying functional improvement needs to be demonstrated. Transplantation approaches on the experimental level need further development before clinical application can be considered.     

Quiescent neural cells regain multipotent stem cell characteristics influenced by adult neural stem cells in co-culture

The source of cells participating in central nervous system (CNS) tissue repair and regeneration is poorly defined. One possible source is quiescent neural cells that can persist in CNS in the form of dormant progenitors or highly specialized cell types. Under appropriate conditions, these quiescent cells may be capable of re-entering the mitotic cell cycle and contributing to the stem cell pool. The aim of this study was to determine whether in vitro differentiated neural stem cells (NSC) can regain their multipotent-like stem cell characteristics in co-culture with NSC. To this end, we induced neural differentiation by plating NSC, derived from the periventricular subependymal zone (SEZ) of ROSA26 transgenic mice in Neurobasal A/B27 medium in the absence of bFGF. Under these conditions, NSC differentiated into neurons, glia, and oligodendrocytes. While the level of Nestin expression was downregulated, persistence of dormant progenitors could not be ruled out. However, further addition of bFGF or bFGF/EGF with conditioned medium derived from adult NSC did not induce any noticeable cell proliferation. In another experiment, differentiated neural cells were cultured with adult NSC, isolated from the hippocampus of Balb/c mice, in the presence bFGF. This resulted in proliferating colonies of ROSA26 derived cells that mimicked NSC in their morphology, growth kinetics, and expressed NSC marker proteins. The average nuclear area and DAPI fluorescence intensity of these cells were similar to that of NSC grown alone. We conclude that reactivation of quiescent neural cells can be initiated by NSC-associated short-range cues but not by cell fusion.     

Clinical and pathological effects of intrathecal injection of mesenchymal stem cell-derived neural progenitors in an experimental model of multiple sclerosis

Multiple sclerosis (MS) is associated with irreversible disability in a significant proportion of patients. At present, there is no treatment to halt or reverse the progression of established disability. In an effort to develop cell therapy-based strategies for progressive MS, we investigated the pre-clinical efficacy of bone marrow mesenchymal stem cell-derived neural progenitors (MSC-NPs) as an autologous source of stem cells. MSC-NPs consist of a subpopulation of bone marrow MSCs with neural progenitor and immunoregulatory properties, and a reduced capacity for mesodermal differentiation, suggesting that this cell population may be appropriate for clinical application in the CNS. We investigated whether MSC-NPs could promote repair and recovery after intrathecal injection into mice with EAE. Multiple injections of MSC-NPs starting at the onset of the chronic phase of disease improved neurological function compared to controls, whereas a single injection had no effect on disease scores. Intrathecal injection of MSC-NPs correlated with reduced immune cell infiltration, reduced area of demyelination, and increased number of endogenous nestin-positive progenitor cells in EAE mice. These observations suggest that MSC-NPs may influence the rate of repair through effects on endogenous progenitors in the spinal cord. This study supports the use of autologous MSC-NPs in MS patients as a means of promoting CNS repair.     

Cell implantation therapies for Parkinson's disease using neural stem, transgenic or xenogeneic donor cells

A new therapeutic neurological and neurosurgical methodology involves cell implantation into the living brain in order to replace intrinsic neuronal systems, that do not spontaneously regenerate after injury, such as the dopaminergic (DA) system affected in Parkinson's disease (PD) and aging. Current clinical data indicate proof of principle for this cell implantation therapy for PD. Furthermore, the disease process does not appear to negatively affect the transplanted cells, although the patient's endogenous DA system degeneration continues. However, the optimal cells for replacement, such as highly specialized human fetal dopaminergic cells capable of repairing an entire degenerated nigro-striatal system, cannot be reliably obtained or generated in sufficient numbers for a standardized medically effective intervention. Xenogeneic and transgenic cell sources of analogous DA cells have shown great utility in animal models and some promise in early pilot studies in PD patients. The cell implantation treatment discipline, using cell fate committed fetal allo- or xenogeneic dopamine neurons and glia, is currently complemented by research on potential stem cell derived DA neurons. Understanding the cell biological principles and developing methodology necessary to generate functional DA progenitors is currently our focus for obtaining DA cells in sufficient quantities for the unmet cell transplantation need for patients with PD and related disorders.