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Central nervous system repair and stem cells
International Congress Series 1302 (2007) 154 – 163
www.ics-elsevier.com
Central nervous system repair and stem cells Rahul Jandial a,b,c,⁎,1 , Ilyas Singec a,c,1 , Vincent J. Duenas a,c , Allen L. Ho a,c , Michael L. Levy d , Evan Y. Snyder a,c a
b
Burnham Institute for Medical Research, Stem Cell & Regeneration Program, 10901 North Torrey Pines Road, La Jolla, CA, 92037, USA University of California San Diego, Department of Surgery, Division of Neurosurgery, 200 West Harbor Drive, San Diego, CA, USA c University of California San Diego, Division of Biological Sciences, San Diego, CA, USA d Children's Hospital San Diego, San Diego, California, USA
Abstract. Stem cells provide us with a future alternative to more traditional pharmacology for treatment of a wide range of pathology that occurs within the central nervous system (CNS). The ability to not only, minimize neuronal and glial degeneration and loss, but also to repair and regenerate the diseases nervous system is currently the investigational horizon for regenerative medicine. For this, neural stem cells (NSCs) that can be derived either from the CNS itself or from pluripotent embryonic stem cells (ESCs), are promising candidates. Their ability to ameliorate disease symptoms and to improve functional recovery has been demonstrated in various animal models of traumatic and ischemic CNS injury and neurodegeneration involving neuronal and glial cells. Further, the possibility of recruiting endogenous stem cells to compliment stem cell transplantation is providing additional promise to the future of stem cell mediated regenerative medicine. © 2007 Published by Elsevier B.V. Keywords: Neural repair; Stem cell; Neural stem cell; Functional recovery; Neural regeneration; Stroke; Spinal cord injury; Parkinson's disease; Stem cell therapeutics
1. Introduction The myriad neurological pathology that can affect the human central nervous system along with the limited self-repair capacity of the CNS, call for new therapeutic strategies. ⁎ Corresponding author. Burnham Institute for Medical Research, Stem Cell & Regeneration Program, 10901 North Torrey Pines Road, La Jolla, CA, 92037, USA. Tel.: +1 858 646 3158 or +1 617 686 5361; fax: +1 858 713 6273. E-mail address: [email protected] (R. Jandial). 1 These authors contributed to this work equally. 0531-5131/ © 2007 Published by Elsevier B.V. doi:10.1016/j.ics.2007.02.062
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Our increasing knowledge about the fundamental biology and therapeutic potential of various stem cell types opened a new chapter in regenerative medicine. The initial work on rodent stem cells over the two last decades is now being successfully continued with stem cells of human origin and from different developmental stages. We have learned about key genes and cellular mechanisms that maintain the stem cells status or lead to differentiated progeny. We have also learned about the multiple roles of stem cells during development, disease, and aging. It is now well-established that stem cells are not only a valuable tool for cell replacement but are equipped with important additional properties that may be harnessed for cell protection, detoxification, and gene therapy. Stem cell biology is being recognized as a continuum of development and developmental processes are tightly regulated, both temporally and spatially. Better understanding of these developmental events is considered to be a key strategy for the successful use of stem cells (endogenous and grafted) for CNS repair and functional recovery. For instance, the generation of functional neurons and glial cells during brain development requires a concerted coordination of cell proliferation, migration, cell-type specification, and synaptic integration all of which are also crucial for successful stem cell therapy [5]. 2. Stem cell prototypes Stem cells give rise to organs and maintain tissue integrity and homeostasis in the adult organism. There are different types of stem cells, including embryonic and somatic (fetal or adult derived) from which new cells can be derived. To fulfill the criteria of a stem cell, as opposed to a “progenitor” cell, a single clonal cell must have the following functional properties: (1) should be able to generate the cell types from the organ it was derived from, and (2) possess “self-renewal”, i.e., the ability to produce daughter cells with identical properties. The ability to populate a developing or injured region with appropriate cell types upon transplantation is another important stem cell feature that is well-established with hematopoetic stem cells and awaits standardization in other organ systems including the brain. In the following we will introduce two prototypical stem cells, the embryonic stem cell (ESC) and neural stem cells (NSC), and discuss their potentials for neural repair [7]. 2.1. Embryonic stem cells Embryonic stem cells (ESCs) have been derived from the inner cell mass of blastocysts of different species including human. They can be totipotent (be able to generate all cells types in an organism except the placenta), pluripotent (the ability to yield mature cell types from all different germ layers), or multipotent (be able to give rise to all cells within an organ). Work performed with mouse ESCs has provided proof-of-principle that pluripotent cell lines can be harnessed for developmental biological studies as well as for new therapeutics. Since significant species differences exist between mouse and human ESCs regarding signalling pathways and molecular regulation of pluripotency, it is pivotal to fully characterize and define the molecular mechanisms in human ESCs. Currently, our understanding of human ESCs cells is increasing and knowledge is being accumulated on improved cell culture conditions, long-term propagation, controlled differentiation, and transplantation into animal models of human disease [12].
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The list of various cell types differentiated from human ESCs (e.g. neurons, cardiomyocytes, hepatocytes) is continuously increasing. Pluripotent ESCs can be stepwise differentiated in the culture dish by recapitulating aspects of in vivo development and the use of relevant epigenetic factors. Importantly, the acquisition of a particular developmental stage of a cell is best characterized by considering morphological, immunophenotypic, and functional criteria. The unlimited access to specific functional human cells is expected to play not only an important role in therapeutic cell replacement but also for disease modelling and drug screening. 2.2. Neural stem cells In contrast to pluripotent ESCs, somatic stem cells are believed to be multipotent thereby capable of generating the major cell types limited to the tissue of origin. Typically, the NSC is capable of producing neurons, astrocytes, and oligodendrocytes. Somatic/tissue-specific stem cells are the building blocks of organs during development and survive in specialized microenvironments (“stem cell niche”) contributing to new cells throughout life. NSCs are (1) multipotent (the ability to yield mature cells in all 3 fundamental neural lineages throughout the nervous system: neurons; astrocytes; and oligodendrocytes), have the (2) ability to populate a developing region and/or repopulate an ablated or degenerated region of the CNS with appropriate cell types, and (3) undergo “self-renewal”, i.e., the ability to produce daughter cells with identical properties. Neural stem cells have so far been identified in vitro. No study has been able to demonstrate the existence of multipotent NSCs in vivo. NSCs are highly abundant during embryogenesis, with a sharp decline shortly after birth. In the adult nervous system, NSCs are confined to the subgranular zone (SGZ) in the dentate gyrus of the hippocampus and the subventricular zone (SVZ) lining the lateral ventricles [1,4]. The newly born neurons in hippocampus have been suggested to improve memory and play a role in mood behavior such as stress and depression. Neuroblasts born in the SVZ migrate along the rostral migratory stream (RMS) to the olfactory bulbs where they differentiate into periglomelular and granule neurons. Isolation of cells from brain regions such as amygdala, substantia nigra, and cortex, have included cells with stem cell-characteristics in vitro. Morphologically, NSCs share properties with both astrocytes and radial glia. The main characteristic is a long process that extends radially. Although no definitive marker have been suggested for neural stem cells, a substantial amount of work shows that they are positive for nestin, an intermediary filament protein, and glial fibrillary acidic protein (GFAP), used traditionally to identify astrocytes [8]. NSCs or progenitor cells with a more restricted developmental potential, can be generated from hESCs or directly isolated from the developing CNS as well as from neurogenic regions of the adult brain. Historically, the first established NSC lines exploited knowledge accumulated on tumor viruses and immortalization. These cell lines have been invaluable in expanding our experience on basic stem cell biology and neural repair. Some of these multipotent cell lines, such as the C17.2 NSC line, are still widely used. However, NSC that have not been genetically modified can also be propagated in vitro for extended periods of time using high concentrations of mitogenic factors such as basis fibroblast growth factor (bFGF) and epidermal growth factor (EGF). Neural stem/progenitor cells have been cultured as monolayers on coated substrates or as free-floating spherical aggregated, termed neurospheres [13].
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3. Stem cell repertoire 3.1. Chaperone effects Initially, stem cells were exclusively considered as tools for cell replacement. However, there is multiple evidence now for robust additional biological properties (“chaperone effects”) of stem cells that may be exploited therapeutically. Chaperone effects of stem cells include the natural delivery of neurotrophic, cytoprotective, and anti-inflammatory molecules (e.g. GDNF, BDNF, NT-3) in order to rescue dysfunctional cells. This concept of stem cellbased chaperone effects was first demonstrated in the brain of aged and parkinsonian mice and later confirmed and extended to other organ systems and various diseases (e.g. bone marrowderived mesenchymal stem cells or embryo. 3.2. Environmental cues Increasingly, the microenvironments within the CNS are providing insight into the molecular milieu regulating stem cell biology. A specialized microenvironment in the neurogenic regions is responsible for the continued self-renewal and differentiation of the stem cell pool. It is also known that the cellular milieu contributes to fate determination and cortical development, specifically through the effects of resident astrocytes in both the subventricular zone (SVZ) and hippocampus. Physical exercise and enriched environment have been shown to promote neurogenesis in the SGZ. The effects from physical activity are partly mediated by IGF-1, VEGF, BDNF and endogenous opioids. The characterization of the stem cell microenvironment will provide the molecular and cellular scaffold upon which stem cell therapy, both endogenous and exogenous, can be built. 3.3. Stem cells as vectors for gene therapy Brain lesions can be focal and restricted to a certain brain region or widely distributed in the parenchyma. Ideally, both lesion types would be targeted with a specific and efficient delivery of therapeutic molecules and drugs. In fact, efficient delivery is still a major hurdle in gene therapy. The finding that endogenous and grafted NSCs display an extensive migratory potential and tropism toward brain lesions founded the idea that these cells may be used as therapeutic vectors. Proof-of-principle experiments in animal models of lysosomal storage diseases (example for a widely distributed brain lesions) and brain tumors (example for a focal lesion) have shown that genetically modified NSCs are powerful therapeutics to cross-correct hereditary enzymatic deficiencies or to dramatically reduce a tumor mass. Thus, NSCs hold great promise for both cell and gene therapy. 4. Stem cell-base CNS repair It is manifest that stem cells can be used to replace neuronal, astrocytic, and oligodendroglial cells lost due to various brain diseases. However, it is important to note that a successful use of stem cell is probably dependent on many factors including, nature and degree of injury, disease history and age of the patient, primarily affected cell types, type of stem cell chosen for
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transplantation, and the site of grafting. A deeper insight into these parameters will be important to tailor patient-specific treatment paradigms in a clinical context [9]. 4.1. Parkinson's disease Parkinson's disease (PD) is characterized by a progressive deterioration and loss of nigrostriatal dopaminergic neurons in the substantia nigra. The consequence of this cell death in the ventral midbrain is a deficient dopamine neurotransmission in the target region, the striatum. Clinically, the PD presents with clinical symptoms such as tremor, rigidity, and bradykinesia. Patients transplanted with fetal mesencephalic grafts in the early 1990s have demonstrated that an ectopic transplantation of dopamine producing cells into the striatum can restore motor function and ameliorate clinical symptoms. Because of the limited availability of fetal tissue, stem cells are expected to provide unlimited numbers of transplantable dopamine neurons. Several studies using rodent and primate models of PD have demonstrated successful integration and functional improvement after grafting of dopaminergic neurons derived from both ESCs and NSCs. In primate models, monkey embryonic stem cells have been transplanted and animals evaluated for behavioral improvements. As with the rodent models functional improvements occur. Furthermore these behavior assessments can be corroborated with functional neuroimaging [15]. (Fig. 1a) The considerable progress in stem cell-based treatment of PD in animals still faces many challenges before clinical translation. Human ESCs differentiate to dopaminergic neurons under various protocols, yet the creation of a purified and homogenous population of dopaminergic neurons is challenging and needs improvement. Animal models for future investigation should increasingly include primates in order to refine the mechanics and logistics of transplantation. Patients in whom stem cell therapy will be the most effective with the least side effects should be defined. The effects of post-transplantation training and rehabilitation need to be better understood, it appears that these contribute to improved functional outcome in experimental animal models. The clinical experience with fetal grafts suggests that the patient's disease history is an important parameter and that cell therapy will fail to be the method of choice for every parkinsonian patient, thereby patient selection will be pivotal for clinical improvements after graft placement. Finally, the adverse side-effects such as dyskinesias observed in some patients after transplantation of fetal grafts need careful consideration, and further the safety hESCs needs to be established prior to clinical transplantation [14]. 4.2. Stroke Arterial occlusion within the brain can lead to ischemia and infarction of brain parenchyma. The current treatment of stroke remains limited, and focuses on neuroprotection to limit the expansion of the infarct and to possibly recover the cells within the ischemic penumbra. The use of recombinant tissue plasminogen activator in select clinical situations within a critical time window after the stroke event has led to improved clinical outcome. Unfortunately, the time constraints in which this treatment can be offered limits potential application to a very small group of stroke patients. Hypoxic/ischemic injury can lead to substantial tissue loss and the formation of infarction cavities which would be a major therapeutic obstacle for the survival of newly seeded stem
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cells. Our group has demonstrated extensively that damaged brain areas can be repaired by the combined use of stem cells together with polymer scaffolds that can be placed into the infarction cavity [11]. (Fig. 1b) It has been suggested that stem cells may be uniquely suited for stroke therapy given their inherent cytoprotective, anti-inflammatory, and restorative properties. Noteably, in early studies using other cellular therapies (e.g. human NT-2 teratocarcinoma line) have demonstrated some functional improvement in stoke patients. However, the growth of stem cells is better to control than any other immortalized cell line. Stem cell-derived and implanted neurons were shown to survive for N 2 years in the human brain. Stem cell use in animal models of cerebral ischemia clearly demonstrates the ability of murine and human NSCs to engraft into the brain and survive, migrate, and specifically differentiate leading to functional outcome. Other studies with murine NSCs have shown the potential of stem cell grafts to promote recovery in ischemic rats, and recovery of sensorimotor deficits after transplantation into the striatum and cortex ipisilateral or contralateral to the stroke. In a clinical setting, cell transplants for stroke patients may be feasible even weeks after the ischemic event, allowing the patient to recover from the acute injury. Furthermore, several weeks may be needed to perform detailed neurophysiological and behavioral testing to allow selection of the candidate patients. In accordance with a timetable that accounts for the most likely clinical scenario with patients, human somatic NSCs have led to functional recovery from stroke with improvement at both cortical and subcortical levels in various murine models of stroke. Embryonic stem cells can be differentiated into NSCs following exposure to retinoic acid in vitro and have also, demonstrated functional recovery in rodents [10].
Fig. 1. a [15]. Function of ES cell-derived neurospheres in MPTP-treated monkeys. Behavioral scores (A) and PET study (B and C) of ES cell-transplanted (n = 6) and sham-operated animals (n = 4). (B) Mean Ki values from entire putamen. (C) Increased 18F-fluorodopa uptake in the putamen of ES cell-transplanted animals. All values are mean ± SD. ⁎p b 0.05. b [11]. Implantation of NSC–PGA complexes into a region of cavity formation following extensive HI brain injury and necrosis (A) Brain of an untransplanted (non-Tx) mouse subjected to right HI injury with extensive infarction and cavitation of the ipsilateral right cortex, striatum, thalamus, and hippocampus (arrow). (B) Contrasting with (A), the brain of a similarly injured mouse implanted with an NSC–PGA complex (PGA + NSCs), into the infarction cavity seven days after the induction of HI (arrow; n = 60). At maturity (age-matched to the animal pictured in (A)), the NSC–scaffold complex appears, in this whole mount, to have filled the cavity (arrow) and become incorporated into the infarcted cerebrum. (C, D) Higher magnification of representative coronal sections through that region, in which parenchyma appears to have filled in spaces between the dissolving black polymer fibers (white arrow in (C)) and even to support neovascularization by host tissues, as seen in (D). A blood vessel is indicated by the closed black arrow in (D); open arrow in (D) points to degrading black polymer fiber.
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Although, regenerative cell therapy for stroke appears very promising, the use of stem cells is in its infancy. Clearly, the mechanisms that led to beneficial effects after stem cell transplantation need to be better understood. For instance, it is important to define whether the reported improvements are primarily the result of reconstitution of neural circuitry by cell replacement, from the enhancement of intrinsic repair mechanisms (including the recruitment of endogenous stem cells), or even both. Most likely, grafted and endogenous stem cells are effective through a multitude of mechanisms. It is possible that stem cells may be delivered to the injured brain not only by local intracerebral delivery, but also by intravenous or intrathecal routes. 4.3. Spinal cord injury Spinal cord injury (SCI) remains a devastating ailment with little opportunity for treatment. The injury occurs from mechanical forces in the acute setting and is exacerbated by secondary inflammatory damage, both leading to neuronal death and demyelination. Accordingly, potential therapy would vary depending on the time frame after injury, with minimizing inflammation the primary concern early after injury and regeneration the major goal when injury is in its chronic phase. The pathobiology of SCI is highly dependent on the time course in which the injured spinal cord is examined. This directs the transplantation of NSCs or their derivatives (e.g. oligodendrocytes for myelination) to carefully account for and maximize the timing of transplantation with consideration cell survival of grafted cells. Studies have addressed some of these issues demonstrating the importance of both timing of transplantation and the role of growth factors in murine models of SCI treated with NSCs. Injured rats received NSC transplants at different time points that would correspond clinically with subacute and chronic SCI, respectively. The administration of growth factors including EGF, bFGF, and platelet derived growth factor (PDGF) resulted in increased numbers of cells grafted into the injured spinal cord, either by enhanced survival or increased proliferation. To determine if NSCs together with growth factors can lead to neurological improvement, animals were evaluated using three independent behavioral tasks and all three behavior tasks showed significant improvements over control mice, with even some long-term improvements [6]. (Fig. 2a) Human CNS fetal-derived stem cells have been shown to survive, engraft, differentiate and improve locomotor skills after traumatic SCI in mice. Contusive spinal cord injury of the thoracic cord was treated with injection of human NSCs. Functional recovery was assessed and shown to be improved. Also, selective ablation of grafted cells with diptheria toxin (murine cells are 100,000 times less sensitive to diptheria toxin than are human cells) was used for the targeted killing of human NSCs. This selective ablation led to reversal of symptomatic and behavioral improvement, providing further support that human NSCs can mediate functional recovery in murine spinal cord injury models. Despite of demonstrating functional recovery in murine models of SCI, with both murine and human NSCs, it remains that for clinical translation more investigation with primate models is critical prior to any human trials. Indeed, spinal cord anatomy is different in the rodent as compared to primates and humans [3]. Neural regeneration is not without pitfalls, as some studies have shown the creation of aberrant axonal sprouting which led to allodynia hypersensitivity. This may be related to excessive astroglial differentiation, highlighting the need for more controlled differentiation in
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Fig. 2. a [6]. Subacute transplantation of YFP-NPCs resulted in a significant locomotor recovery compared with injured rats in the control group. A, BBB rating scale showed a significant improvement in the locomotor BBB score in transplanted rats at 3 weeks after transplantation compared with the plain injured and control groups (n = 5 for plain injured group and n = 8 for other groups). B, Using grid-walk analysis, transplanted rats also showed fewer errors in hindlimb placements at 5 and 6 weeks after transplantation compared with the plain injured and control groups (n = 5 for plain injured group and n = 8 for other groups). C, Representative footprints of normal, plain injured, control, and grafted rats (n = 5 for plain injured group and n = 8 for other groups) shows improvement in interlimb coordination as well as angle of rotation in the transplanted group compared with the plain injured and control groups. D, E, Footprint analysis revealed that transplantation with adult NPCs significantly improved interlimb coordination and reduced the hindlimb angle of rotation at 5 and 6 weeks after transplantation. The data show the mean ± SEM. ⁎p b 0.05. b [2]. A subset of newborn layer V cortical neurons extends axons to the cervical spinal cord. (A) Both newborn and original layer V CSMN were retrogradely labeled by FG (blue). (B) Field expanded in C–F showing a BrdUrd+/NeuN+/FG+ triple-labeled adult-born neuron (arrow). (Bar, 10 μm) (C–E) Individual images show that this BrdUrd+ nucleus (C; red) is located within this neuron, which is retrogradely labeled with FG from the cervical spinal cord (D; blue) and expresses NeuN (E; green). (Bar, 10ìm.) (F) Overlay showing BrdUrd+/FG+/NeuN+ neuron colocalization. (G) Higher-magnification overlay of the same neuron from C–F. (H) A separate example of an adult-born neuron with a projection to the spinal cord. Laser-scanning confocal images were combined to produce 3D reconstructions of newborn neurons. Viewing a BrdUrd+/FG+ newborn neuron along its x (H′), y (H″), and z axes (H) unequivocally demonstrates the colocalization of BrdUrd and FG. (I) Quantification of BrdUrd+/FG+ adult-born CSMN extending spinal projections from 12 to 56 weeks after induction of original CSMN apoptosis. Each point indicates the number of adult-born BrdUrd+/FG+ neurons per mm3 in an individual animal; each bar indicates the mean. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
order to both maximize functional recovery and minimize side effects. Along with increased understanding of cellular and molecular mechanisms, the timing and logistics of transplantation need to be improved as well as non-invasive cellular imaging established.
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5. Endogenous stem cell recruitment for CNS repair The alternative or compliment to stem cell transplantation would be the manipulation of endogenous stem cells for therapeutic purposes. The advantages would include using the patient's own cells, not needing an invasive procedure, and obviating the concern over the immunogenicity of transplanted cells. It appears that adult neurogenesis is restricted to the olfactory bulb and dentate gyrus of the hippocampus, yet it is possible that some NSCs exist along the entire adult neuraxis [5]. Exploiting endogenous NSCs would require successful coordination of cell proliferation, differentiation, migration and integration, and it does appear that some instructive signals remain in the adult CNS. However, most CNS regions are not permissive for neurogenesis under normal in vivo conditions, yet some studies suggest that endogenous NSCs are primed to respond to environmental signals that exist primarily during pathological states. Accordingly, one approach to accentuate endogenous stem cell proliferation is with the administration of growth factors. Intra-ventricular infusion of transforming growth factor alpha (TGFalpha) into rodents with lesions of the substantia nigra dopaminergic neurons has led to functional improvements, putatively through recruitment of endogenous stem cells. Other likely bioactive molecules with potential to evoke a proliferative response, in regions of the brain with multipotent cells, are neurotrophins. Physiologically, neurotrophins are involved in cell cycle regulation, cell survival, and differentiation and are critical during normal development. Growth factor infusion can also promote proliferation of SVZ derived progenitor cells that gave rise to hippocampal CA1 pyramidal neurons in rodent stroke models, with improvements in spatial orientation. Whether the neurogenic response creates neurons with long-term viability remains to be shown. Another candidate with efficacy in stroke models is erythropoietin (EPO), which has been shown to induce neurogenesis and functional improvement in rats. Furthermore, endogenous neural precursors can differentiate into new neurons that extend long-distance projections to the spinal cord, in the adult rodent. Targeted apoptosis of corticospinal motor neurons was induced and it was demonstrated that adult-born corticospinal motor neurons were generated extending from the motor cortex to the spinal cord [2] (Fig. 2b). The horizon for neural repair includes continued investigation into whether the diseased CNS can be treated with growth and differentiation factors to induce neural repair. As more is learned about the molecular signals and environmental cues, endogenous stem cells may prove to be a compliment or even replacement to transplantation of exogenous stem cells. 6. Other stem cells The use of embryonic or somatic stem cells for brain repair is currently in the focus of rigorous scientific investigation. Other stem cells have also been suggested as sources for cell therapy. For instance, some groups have found that mesenchymal stem cells (MSCs) can differentiate into astrocytes and neurons in vitro and in vivo, and may have the advantage over ESCs or NSCs by being highly accessible source for the patient's own stem cells. However, there is ongoing controversy about the plasticity and developmental potential of MSCs. Some groups suggested that the findings made with MSCs may be cell culture artifacts rather than being true differentiation into unexpected cell types. Therefore, it is crucial to assay the differentiation of any stem cell into a particular cell type by
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combining morphological, immunophenotypic, and functional criteria. Currently, MSCs do not appear as a realistic alternative for the use of ESCs or NSCs for neural repair [5]. 7. Conclusion Stem cell biology represents a strong foundation for neural repair. So far gained experimental evidence suggests that this technology may be applicable to treat patients in the future. Since ESCs can be multiplied indefinitely and have the potential to give rise to a variety of functional human cells, it is conceivable to believe that stem cells will play an important role in disease modelling and drug testing. Moreover, since stem cells mimic aspects of normal development, these cells may be used to study early steps of human development which would not be accessible for experimentation otherwise. We have highlighted current problems in the rapidly progressing stem cell field which involve safety issues, standardization of the protocols used, development of rigorous assays for characterization, accumulation of experimental data in primate models of human disease. Realistic candidate diseases and patients that may benefit from stem cell therapy need to be defined before any clinical application. Since clinicians and stem cell biologists share a strong common interest to understand and treat human disease, stem cells have the true potential to transform modern medicine. References [1] J. Altman, G.D. Das, Autoradiographic and histological evidence of postnatal neurogenesis in rats, J. Comp. Neurol. 124 (1965) 319–335. [2] J. Chen, et al., Neurogenesis of corticospinal motor neurons extending spinal projection in adult mice, PNAS 46 (2004) 16357–16362. [3] B.J. Cummings, et al., Human neural stem cells differentiate and promote locomoter recovery in spinal cordinjured mice, PNAS 102 (2005) 14069–14074. [4] A. Falk, J. Frisen, New neurons in old brains, Ann. Med. 37 (2005) 480–486. [5] S. Gilbert, Developmental Biology, 7th ed. Sinauer Associates Publishers, Massachusetts, 2003. [6] S. Karimi-Abdolrezaee, et al., Delayed transplantation of adult neural precursor cells promotes remyelination and functional neurological recovery after spinal cord injury, J. Neurosci. 26 (2006) 3377–3389. [7] R. Lanza, et al., Handbook of Stem Cells, Elsevier Academic Press, Oxford, 2004. [8] R. Lanza, J. Gearhart, B. Hogan, Essentials of Stem Cell Biology, Elsevier Academic Press, Oxford, 2006. [9] O.L. Lindvall, Z. Kokaia, A. Martinez-Serrano, Stem cell therapy for human disorders — how to make it work, Nat. Med. 10 (2004) S42–S50. [10] H. Nakatomi, et al., Regeneration of hippocampal pyramidal neurons after ischemic brain injury by recruitment of endogenous neural progenitors, Cell 110 (2002) 429–441. [11] K.I. Park, Y.D. Teng, E.Y. Snyder, The injured brain interacts reciprocally with neural stem cells supported by scaffolds to reconstitute lost tissue, Nat. Biotechnol. 20 (2002) 1111–1117. [12] B.A. Reyonals, S. Weiss, Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell, Dev. Biol. 175 (1996) 1–13. [13] I. Singec, et al., Defining the actual sensitivity and specificity of the neurosphere assay in stem cell biology, Nat. Methods 3 (2006) 801–806. [14] K. Soderstrom, et al., Neural repair strategies for Parkinson's disease: insights from primate models, Cell Transplant 15 (2006) 251–265. [15] Y. Takagi, et al., Dopaminergic neurons generated from monkey embryonic stem cells function in a Parkinson primate model, J. Clin. Invest. 115 (2005) 102–109.
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Central nervous system repair and stem cells Rahul Jandial a,b,c,⁎,1 , Ilyas Singec a,c,1 , Vincent J. Duenas a,c , Allen L. Ho a,c , Michael L. Levy d , Evan Y. Snyder a,c a
b
Burnham Institute for Medical Research, Stem Cell & Regeneration Program, 10901 North Torrey Pines Road, La Jolla, CA, 92037, USA University of California San Diego, Department of Surgery, Division of Neurosurgery, 200 West Harbor Drive, San Diego, CA, USA c University of California San Diego, Division of Biological Sciences, San Diego, CA, USA d Children's Hospital San Diego, San Diego, California, USA
Abstract. Stem cells provide us with a future alternative to more traditional pharmacology for treatment of a wide range of pathology that occurs within the central nervous system (CNS). The ability to not only, minimize neuronal and glial degeneration and loss, but also to repair and regenerate the diseases nervous system is currently the investigational horizon for regenerative medicine. For this, neural stem cells (NSCs) that can be derived either from the CNS itself or from pluripotent embryonic stem cells (ESCs), are promising candidates. Their ability to ameliorate disease symptoms and to improve functional recovery has been demonstrated in various animal models of traumatic and ischemic CNS injury and neurodegeneration involving neuronal and glial cells. Further, the possibility of recruiting endogenous stem cells to compliment stem cell transplantation is providing additional promise to the future of stem cell mediated regenerative medicine. © 2007 Published by Elsevier B.V. Keywords: Neural repair; Stem cell; Neural stem cell; Functional recovery; Neural regeneration; Stroke; Spinal cord injury; Parkinson's disease; Stem cell therapeutics
1. Introduction The myriad neurological pathology that can affect the human central nervous system along with the limited self-repair capacity of the CNS, call for new therapeutic strategies. ⁎ Corresponding author. Burnham Institute for Medical Research, Stem Cell & Regeneration Program, 10901 North Torrey Pines Road, La Jolla, CA, 92037, USA. Tel.: +1 858 646 3158 or +1 617 686 5361; fax: +1 858 713 6273. E-mail address: [email protected] (R. Jandial). 1 These authors contributed to this work equally. 0531-5131/ © 2007 Published by Elsevier B.V. doi:10.1016/j.ics.2007.02.062
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Our increasing knowledge about the fundamental biology and therapeutic potential of various stem cell types opened a new chapter in regenerative medicine. The initial work on rodent stem cells over the two last decades is now being successfully continued with stem cells of human origin and from different developmental stages. We have learned about key genes and cellular mechanisms that maintain the stem cells status or lead to differentiated progeny. We have also learned about the multiple roles of stem cells during development, disease, and aging. It is now well-established that stem cells are not only a valuable tool for cell replacement but are equipped with important additional properties that may be harnessed for cell protection, detoxification, and gene therapy. Stem cell biology is being recognized as a continuum of development and developmental processes are tightly regulated, both temporally and spatially. Better understanding of these developmental events is considered to be a key strategy for the successful use of stem cells (endogenous and grafted) for CNS repair and functional recovery. For instance, the generation of functional neurons and glial cells during brain development requires a concerted coordination of cell proliferation, migration, cell-type specification, and synaptic integration all of which are also crucial for successful stem cell therapy [5]. 2. Stem cell prototypes Stem cells give rise to organs and maintain tissue integrity and homeostasis in the adult organism. There are different types of stem cells, including embryonic and somatic (fetal or adult derived) from which new cells can be derived. To fulfill the criteria of a stem cell, as opposed to a “progenitor” cell, a single clonal cell must have the following functional properties: (1) should be able to generate the cell types from the organ it was derived from, and (2) possess “self-renewal”, i.e., the ability to produce daughter cells with identical properties. The ability to populate a developing or injured region with appropriate cell types upon transplantation is another important stem cell feature that is well-established with hematopoetic stem cells and awaits standardization in other organ systems including the brain. In the following we will introduce two prototypical stem cells, the embryonic stem cell (ESC) and neural stem cells (NSC), and discuss their potentials for neural repair [7]. 2.1. Embryonic stem cells Embryonic stem cells (ESCs) have been derived from the inner cell mass of blastocysts of different species including human. They can be totipotent (be able to generate all cells types in an organism except the placenta), pluripotent (the ability to yield mature cell types from all different germ layers), or multipotent (be able to give rise to all cells within an organ). Work performed with mouse ESCs has provided proof-of-principle that pluripotent cell lines can be harnessed for developmental biological studies as well as for new therapeutics. Since significant species differences exist between mouse and human ESCs regarding signalling pathways and molecular regulation of pluripotency, it is pivotal to fully characterize and define the molecular mechanisms in human ESCs. Currently, our understanding of human ESCs cells is increasing and knowledge is being accumulated on improved cell culture conditions, long-term propagation, controlled differentiation, and transplantation into animal models of human disease [12].
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The list of various cell types differentiated from human ESCs (e.g. neurons, cardiomyocytes, hepatocytes) is continuously increasing. Pluripotent ESCs can be stepwise differentiated in the culture dish by recapitulating aspects of in vivo development and the use of relevant epigenetic factors. Importantly, the acquisition of a particular developmental stage of a cell is best characterized by considering morphological, immunophenotypic, and functional criteria. The unlimited access to specific functional human cells is expected to play not only an important role in therapeutic cell replacement but also for disease modelling and drug screening. 2.2. Neural stem cells In contrast to pluripotent ESCs, somatic stem cells are believed to be multipotent thereby capable of generating the major cell types limited to the tissue of origin. Typically, the NSC is capable of producing neurons, astrocytes, and oligodendrocytes. Somatic/tissue-specific stem cells are the building blocks of organs during development and survive in specialized microenvironments (“stem cell niche”) contributing to new cells throughout life. NSCs are (1) multipotent (the ability to yield mature cells in all 3 fundamental neural lineages throughout the nervous system: neurons; astrocytes; and oligodendrocytes), have the (2) ability to populate a developing region and/or repopulate an ablated or degenerated region of the CNS with appropriate cell types, and (3) undergo “self-renewal”, i.e., the ability to produce daughter cells with identical properties. Neural stem cells have so far been identified in vitro. No study has been able to demonstrate the existence of multipotent NSCs in vivo. NSCs are highly abundant during embryogenesis, with a sharp decline shortly after birth. In the adult nervous system, NSCs are confined to the subgranular zone (SGZ) in the dentate gyrus of the hippocampus and the subventricular zone (SVZ) lining the lateral ventricles [1,4]. The newly born neurons in hippocampus have been suggested to improve memory and play a role in mood behavior such as stress and depression. Neuroblasts born in the SVZ migrate along the rostral migratory stream (RMS) to the olfactory bulbs where they differentiate into periglomelular and granule neurons. Isolation of cells from brain regions such as amygdala, substantia nigra, and cortex, have included cells with stem cell-characteristics in vitro. Morphologically, NSCs share properties with both astrocytes and radial glia. The main characteristic is a long process that extends radially. Although no definitive marker have been suggested for neural stem cells, a substantial amount of work shows that they are positive for nestin, an intermediary filament protein, and glial fibrillary acidic protein (GFAP), used traditionally to identify astrocytes [8]. NSCs or progenitor cells with a more restricted developmental potential, can be generated from hESCs or directly isolated from the developing CNS as well as from neurogenic regions of the adult brain. Historically, the first established NSC lines exploited knowledge accumulated on tumor viruses and immortalization. These cell lines have been invaluable in expanding our experience on basic stem cell biology and neural repair. Some of these multipotent cell lines, such as the C17.2 NSC line, are still widely used. However, NSC that have not been genetically modified can also be propagated in vitro for extended periods of time using high concentrations of mitogenic factors such as basis fibroblast growth factor (bFGF) and epidermal growth factor (EGF). Neural stem/progenitor cells have been cultured as monolayers on coated substrates or as free-floating spherical aggregated, termed neurospheres [13].
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3. Stem cell repertoire 3.1. Chaperone effects Initially, stem cells were exclusively considered as tools for cell replacement. However, there is multiple evidence now for robust additional biological properties (“chaperone effects”) of stem cells that may be exploited therapeutically. Chaperone effects of stem cells include the natural delivery of neurotrophic, cytoprotective, and anti-inflammatory molecules (e.g. GDNF, BDNF, NT-3) in order to rescue dysfunctional cells. This concept of stem cellbased chaperone effects was first demonstrated in the brain of aged and parkinsonian mice and later confirmed and extended to other organ systems and various diseases (e.g. bone marrowderived mesenchymal stem cells or embryo. 3.2. Environmental cues Increasingly, the microenvironments within the CNS are providing insight into the molecular milieu regulating stem cell biology. A specialized microenvironment in the neurogenic regions is responsible for the continued self-renewal and differentiation of the stem cell pool. It is also known that the cellular milieu contributes to fate determination and cortical development, specifically through the effects of resident astrocytes in both the subventricular zone (SVZ) and hippocampus. Physical exercise and enriched environment have been shown to promote neurogenesis in the SGZ. The effects from physical activity are partly mediated by IGF-1, VEGF, BDNF and endogenous opioids. The characterization of the stem cell microenvironment will provide the molecular and cellular scaffold upon which stem cell therapy, both endogenous and exogenous, can be built. 3.3. Stem cells as vectors for gene therapy Brain lesions can be focal and restricted to a certain brain region or widely distributed in the parenchyma. Ideally, both lesion types would be targeted with a specific and efficient delivery of therapeutic molecules and drugs. In fact, efficient delivery is still a major hurdle in gene therapy. The finding that endogenous and grafted NSCs display an extensive migratory potential and tropism toward brain lesions founded the idea that these cells may be used as therapeutic vectors. Proof-of-principle experiments in animal models of lysosomal storage diseases (example for a widely distributed brain lesions) and brain tumors (example for a focal lesion) have shown that genetically modified NSCs are powerful therapeutics to cross-correct hereditary enzymatic deficiencies or to dramatically reduce a tumor mass. Thus, NSCs hold great promise for both cell and gene therapy. 4. Stem cell-base CNS repair It is manifest that stem cells can be used to replace neuronal, astrocytic, and oligodendroglial cells lost due to various brain diseases. However, it is important to note that a successful use of stem cell is probably dependent on many factors including, nature and degree of injury, disease history and age of the patient, primarily affected cell types, type of stem cell chosen for
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transplantation, and the site of grafting. A deeper insight into these parameters will be important to tailor patient-specific treatment paradigms in a clinical context [9]. 4.1. Parkinson's disease Parkinson's disease (PD) is characterized by a progressive deterioration and loss of nigrostriatal dopaminergic neurons in the substantia nigra. The consequence of this cell death in the ventral midbrain is a deficient dopamine neurotransmission in the target region, the striatum. Clinically, the PD presents with clinical symptoms such as tremor, rigidity, and bradykinesia. Patients transplanted with fetal mesencephalic grafts in the early 1990s have demonstrated that an ectopic transplantation of dopamine producing cells into the striatum can restore motor function and ameliorate clinical symptoms. Because of the limited availability of fetal tissue, stem cells are expected to provide unlimited numbers of transplantable dopamine neurons. Several studies using rodent and primate models of PD have demonstrated successful integration and functional improvement after grafting of dopaminergic neurons derived from both ESCs and NSCs. In primate models, monkey embryonic stem cells have been transplanted and animals evaluated for behavioral improvements. As with the rodent models functional improvements occur. Furthermore these behavior assessments can be corroborated with functional neuroimaging [15]. (Fig. 1a) The considerable progress in stem cell-based treatment of PD in animals still faces many challenges before clinical translation. Human ESCs differentiate to dopaminergic neurons under various protocols, yet the creation of a purified and homogenous population of dopaminergic neurons is challenging and needs improvement. Animal models for future investigation should increasingly include primates in order to refine the mechanics and logistics of transplantation. Patients in whom stem cell therapy will be the most effective with the least side effects should be defined. The effects of post-transplantation training and rehabilitation need to be better understood, it appears that these contribute to improved functional outcome in experimental animal models. The clinical experience with fetal grafts suggests that the patient's disease history is an important parameter and that cell therapy will fail to be the method of choice for every parkinsonian patient, thereby patient selection will be pivotal for clinical improvements after graft placement. Finally, the adverse side-effects such as dyskinesias observed in some patients after transplantation of fetal grafts need careful consideration, and further the safety hESCs needs to be established prior to clinical transplantation [14]. 4.2. Stroke Arterial occlusion within the brain can lead to ischemia and infarction of brain parenchyma. The current treatment of stroke remains limited, and focuses on neuroprotection to limit the expansion of the infarct and to possibly recover the cells within the ischemic penumbra. The use of recombinant tissue plasminogen activator in select clinical situations within a critical time window after the stroke event has led to improved clinical outcome. Unfortunately, the time constraints in which this treatment can be offered limits potential application to a very small group of stroke patients. Hypoxic/ischemic injury can lead to substantial tissue loss and the formation of infarction cavities which would be a major therapeutic obstacle for the survival of newly seeded stem
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cells. Our group has demonstrated extensively that damaged brain areas can be repaired by the combined use of stem cells together with polymer scaffolds that can be placed into the infarction cavity [11]. (Fig. 1b) It has been suggested that stem cells may be uniquely suited for stroke therapy given their inherent cytoprotective, anti-inflammatory, and restorative properties. Noteably, in early studies using other cellular therapies (e.g. human NT-2 teratocarcinoma line) have demonstrated some functional improvement in stoke patients. However, the growth of stem cells is better to control than any other immortalized cell line. Stem cell-derived and implanted neurons were shown to survive for N 2 years in the human brain. Stem cell use in animal models of cerebral ischemia clearly demonstrates the ability of murine and human NSCs to engraft into the brain and survive, migrate, and specifically differentiate leading to functional outcome. Other studies with murine NSCs have shown the potential of stem cell grafts to promote recovery in ischemic rats, and recovery of sensorimotor deficits after transplantation into the striatum and cortex ipisilateral or contralateral to the stroke. In a clinical setting, cell transplants for stroke patients may be feasible even weeks after the ischemic event, allowing the patient to recover from the acute injury. Furthermore, several weeks may be needed to perform detailed neurophysiological and behavioral testing to allow selection of the candidate patients. In accordance with a timetable that accounts for the most likely clinical scenario with patients, human somatic NSCs have led to functional recovery from stroke with improvement at both cortical and subcortical levels in various murine models of stroke. Embryonic stem cells can be differentiated into NSCs following exposure to retinoic acid in vitro and have also, demonstrated functional recovery in rodents [10].
Fig. 1. a [15]. Function of ES cell-derived neurospheres in MPTP-treated monkeys. Behavioral scores (A) and PET study (B and C) of ES cell-transplanted (n = 6) and sham-operated animals (n = 4). (B) Mean Ki values from entire putamen. (C) Increased 18F-fluorodopa uptake in the putamen of ES cell-transplanted animals. All values are mean ± SD. ⁎p b 0.05. b [11]. Implantation of NSC–PGA complexes into a region of cavity formation following extensive HI brain injury and necrosis (A) Brain of an untransplanted (non-Tx) mouse subjected to right HI injury with extensive infarction and cavitation of the ipsilateral right cortex, striatum, thalamus, and hippocampus (arrow). (B) Contrasting with (A), the brain of a similarly injured mouse implanted with an NSC–PGA complex (PGA + NSCs), into the infarction cavity seven days after the induction of HI (arrow; n = 60). At maturity (age-matched to the animal pictured in (A)), the NSC–scaffold complex appears, in this whole mount, to have filled the cavity (arrow) and become incorporated into the infarcted cerebrum. (C, D) Higher magnification of representative coronal sections through that region, in which parenchyma appears to have filled in spaces between the dissolving black polymer fibers (white arrow in (C)) and even to support neovascularization by host tissues, as seen in (D). A blood vessel is indicated by the closed black arrow in (D); open arrow in (D) points to degrading black polymer fiber.
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Although, regenerative cell therapy for stroke appears very promising, the use of stem cells is in its infancy. Clearly, the mechanisms that led to beneficial effects after stem cell transplantation need to be better understood. For instance, it is important to define whether the reported improvements are primarily the result of reconstitution of neural circuitry by cell replacement, from the enhancement of intrinsic repair mechanisms (including the recruitment of endogenous stem cells), or even both. Most likely, grafted and endogenous stem cells are effective through a multitude of mechanisms. It is possible that stem cells may be delivered to the injured brain not only by local intracerebral delivery, but also by intravenous or intrathecal routes. 4.3. Spinal cord injury Spinal cord injury (SCI) remains a devastating ailment with little opportunity for treatment. The injury occurs from mechanical forces in the acute setting and is exacerbated by secondary inflammatory damage, both leading to neuronal death and demyelination. Accordingly, potential therapy would vary depending on the time frame after injury, with minimizing inflammation the primary concern early after injury and regeneration the major goal when injury is in its chronic phase. The pathobiology of SCI is highly dependent on the time course in which the injured spinal cord is examined. This directs the transplantation of NSCs or their derivatives (e.g. oligodendrocytes for myelination) to carefully account for and maximize the timing of transplantation with consideration cell survival of grafted cells. Studies have addressed some of these issues demonstrating the importance of both timing of transplantation and the role of growth factors in murine models of SCI treated with NSCs. Injured rats received NSC transplants at different time points that would correspond clinically with subacute and chronic SCI, respectively. The administration of growth factors including EGF, bFGF, and platelet derived growth factor (PDGF) resulted in increased numbers of cells grafted into the injured spinal cord, either by enhanced survival or increased proliferation. To determine if NSCs together with growth factors can lead to neurological improvement, animals were evaluated using three independent behavioral tasks and all three behavior tasks showed significant improvements over control mice, with even some long-term improvements [6]. (Fig. 2a) Human CNS fetal-derived stem cells have been shown to survive, engraft, differentiate and improve locomotor skills after traumatic SCI in mice. Contusive spinal cord injury of the thoracic cord was treated with injection of human NSCs. Functional recovery was assessed and shown to be improved. Also, selective ablation of grafted cells with diptheria toxin (murine cells are 100,000 times less sensitive to diptheria toxin than are human cells) was used for the targeted killing of human NSCs. This selective ablation led to reversal of symptomatic and behavioral improvement, providing further support that human NSCs can mediate functional recovery in murine spinal cord injury models. Despite of demonstrating functional recovery in murine models of SCI, with both murine and human NSCs, it remains that for clinical translation more investigation with primate models is critical prior to any human trials. Indeed, spinal cord anatomy is different in the rodent as compared to primates and humans [3]. Neural regeneration is not without pitfalls, as some studies have shown the creation of aberrant axonal sprouting which led to allodynia hypersensitivity. This may be related to excessive astroglial differentiation, highlighting the need for more controlled differentiation in
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Fig. 2. a [6]. Subacute transplantation of YFP-NPCs resulted in a significant locomotor recovery compared with injured rats in the control group. A, BBB rating scale showed a significant improvement in the locomotor BBB score in transplanted rats at 3 weeks after transplantation compared with the plain injured and control groups (n = 5 for plain injured group and n = 8 for other groups). B, Using grid-walk analysis, transplanted rats also showed fewer errors in hindlimb placements at 5 and 6 weeks after transplantation compared with the plain injured and control groups (n = 5 for plain injured group and n = 8 for other groups). C, Representative footprints of normal, plain injured, control, and grafted rats (n = 5 for plain injured group and n = 8 for other groups) shows improvement in interlimb coordination as well as angle of rotation in the transplanted group compared with the plain injured and control groups. D, E, Footprint analysis revealed that transplantation with adult NPCs significantly improved interlimb coordination and reduced the hindlimb angle of rotation at 5 and 6 weeks after transplantation. The data show the mean ± SEM. ⁎p b 0.05. b [2]. A subset of newborn layer V cortical neurons extends axons to the cervical spinal cord. (A) Both newborn and original layer V CSMN were retrogradely labeled by FG (blue). (B) Field expanded in C–F showing a BrdUrd+/NeuN+/FG+ triple-labeled adult-born neuron (arrow). (Bar, 10 μm) (C–E) Individual images show that this BrdUrd+ nucleus (C; red) is located within this neuron, which is retrogradely labeled with FG from the cervical spinal cord (D; blue) and expresses NeuN (E; green). (Bar, 10ìm.) (F) Overlay showing BrdUrd+/FG+/NeuN+ neuron colocalization. (G) Higher-magnification overlay of the same neuron from C–F. (H) A separate example of an adult-born neuron with a projection to the spinal cord. Laser-scanning confocal images were combined to produce 3D reconstructions of newborn neurons. Viewing a BrdUrd+/FG+ newborn neuron along its x (H′), y (H″), and z axes (H) unequivocally demonstrates the colocalization of BrdUrd and FG. (I) Quantification of BrdUrd+/FG+ adult-born CSMN extending spinal projections from 12 to 56 weeks after induction of original CSMN apoptosis. Each point indicates the number of adult-born BrdUrd+/FG+ neurons per mm3 in an individual animal; each bar indicates the mean. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
order to both maximize functional recovery and minimize side effects. Along with increased understanding of cellular and molecular mechanisms, the timing and logistics of transplantation need to be improved as well as non-invasive cellular imaging established.
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5. Endogenous stem cell recruitment for CNS repair The alternative or compliment to stem cell transplantation would be the manipulation of endogenous stem cells for therapeutic purposes. The advantages would include using the patient's own cells, not needing an invasive procedure, and obviating the concern over the immunogenicity of transplanted cells. It appears that adult neurogenesis is restricted to the olfactory bulb and dentate gyrus of the hippocampus, yet it is possible that some NSCs exist along the entire adult neuraxis [5]. Exploiting endogenous NSCs would require successful coordination of cell proliferation, differentiation, migration and integration, and it does appear that some instructive signals remain in the adult CNS. However, most CNS regions are not permissive for neurogenesis under normal in vivo conditions, yet some studies suggest that endogenous NSCs are primed to respond to environmental signals that exist primarily during pathological states. Accordingly, one approach to accentuate endogenous stem cell proliferation is with the administration of growth factors. Intra-ventricular infusion of transforming growth factor alpha (TGFalpha) into rodents with lesions of the substantia nigra dopaminergic neurons has led to functional improvements, putatively through recruitment of endogenous stem cells. Other likely bioactive molecules with potential to evoke a proliferative response, in regions of the brain with multipotent cells, are neurotrophins. Physiologically, neurotrophins are involved in cell cycle regulation, cell survival, and differentiation and are critical during normal development. Growth factor infusion can also promote proliferation of SVZ derived progenitor cells that gave rise to hippocampal CA1 pyramidal neurons in rodent stroke models, with improvements in spatial orientation. Whether the neurogenic response creates neurons with long-term viability remains to be shown. Another candidate with efficacy in stroke models is erythropoietin (EPO), which has been shown to induce neurogenesis and functional improvement in rats. Furthermore, endogenous neural precursors can differentiate into new neurons that extend long-distance projections to the spinal cord, in the adult rodent. Targeted apoptosis of corticospinal motor neurons was induced and it was demonstrated that adult-born corticospinal motor neurons were generated extending from the motor cortex to the spinal cord [2] (Fig. 2b). The horizon for neural repair includes continued investigation into whether the diseased CNS can be treated with growth and differentiation factors to induce neural repair. As more is learned about the molecular signals and environmental cues, endogenous stem cells may prove to be a compliment or even replacement to transplantation of exogenous stem cells. 6. Other stem cells The use of embryonic or somatic stem cells for brain repair is currently in the focus of rigorous scientific investigation. Other stem cells have also been suggested as sources for cell therapy. For instance, some groups have found that mesenchymal stem cells (MSCs) can differentiate into astrocytes and neurons in vitro and in vivo, and may have the advantage over ESCs or NSCs by being highly accessible source for the patient's own stem cells. However, there is ongoing controversy about the plasticity and developmental potential of MSCs. Some groups suggested that the findings made with MSCs may be cell culture artifacts rather than being true differentiation into unexpected cell types. Therefore, it is crucial to assay the differentiation of any stem cell into a particular cell type by
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combining morphological, immunophenotypic, and functional criteria. Currently, MSCs do not appear as a realistic alternative for the use of ESCs or NSCs for neural repair [5]. 7. Conclusion Stem cell biology represents a strong foundation for neural repair. So far gained experimental evidence suggests that this technology may be applicable to treat patients in the future. Since ESCs can be multiplied indefinitely and have the potential to give rise to a variety of functional human cells, it is conceivable to believe that stem cells will play an important role in disease modelling and drug testing. Moreover, since stem cells mimic aspects of normal development, these cells may be used to study early steps of human development which would not be accessible for experimentation otherwise. We have highlighted current problems in the rapidly progressing stem cell field which involve safety issues, standardization of the protocols used, development of rigorous assays for characterization, accumulation of experimental data in primate models of human disease. Realistic candidate diseases and patients that may benefit from stem cell therapy need to be defined before any clinical application. Since clinicians and stem cell biologists share a strong common interest to understand and treat human disease, stem cells have the true potential to transform modern medicine. References [1] J. Altman, G.D. Das, Autoradiographic and histological evidence of postnatal neurogenesis in rats, J. Comp. Neurol. 124 (1965) 319–335. [2] J. Chen, et al., Neurogenesis of corticospinal motor neurons extending spinal projection in adult mice, PNAS 46 (2004) 16357–16362. [3] B.J. Cummings, et al., Human neural stem cells differentiate and promote locomoter recovery in spinal cordinjured mice, PNAS 102 (2005) 14069–14074. [4] A. Falk, J. Frisen, New neurons in old brains, Ann. Med. 37 (2005) 480–486. [5] S. Gilbert, Developmental Biology, 7th ed. Sinauer Associates Publishers, Massachusetts, 2003. [6] S. Karimi-Abdolrezaee, et al., Delayed transplantation of adult neural precursor cells promotes remyelination and functional neurological recovery after spinal cord injury, J. Neurosci. 26 (2006) 3377–3389. [7] R. Lanza, et al., Handbook of Stem Cells, Elsevier Academic Press, Oxford, 2004. [8] R. Lanza, J. Gearhart, B. Hogan, Essentials of Stem Cell Biology, Elsevier Academic Press, Oxford, 2006. [9] O.L. Lindvall, Z. Kokaia, A. Martinez-Serrano, Stem cell therapy for human disorders — how to make it work, Nat. Med. 10 (2004) S42–S50. [10] H. Nakatomi, et al., Regeneration of hippocampal pyramidal neurons after ischemic brain injury by recruitment of endogenous neural progenitors, Cell 110 (2002) 429–441. [11] K.I. Park, Y.D. Teng, E.Y. Snyder, The injured brain interacts reciprocally with neural stem cells supported by scaffolds to reconstitute lost tissue, Nat. Biotechnol. 20 (2002) 1111–1117. [12] B.A. Reyonals, S. Weiss, Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell, Dev. Biol. 175 (1996) 1–13. [13] I. Singec, et al., Defining the actual sensitivity and specificity of the neurosphere assay in stem cell biology, Nat. Methods 3 (2006) 801–806. [14] K. Soderstrom, et al., Neural repair strategies for Parkinson's disease: insights from primate models, Cell Transplant 15 (2006) 251–265. [15] Y. Takagi, et al., Dopaminergic neurons generated from monkey embryonic stem cells function in a Parkinson primate model, J. Clin. Invest. 115 (2005) 102–109.