NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.

Noebels JL, Avoli M, Rogawski MA, et al., editors. Jasper's Basic Mechanisms of the Epilepsies [Internet]. 4th edition. Bethesda (MD): National Center for Biotechnology Information (US); 2012.

Cover of Jasper's Basic Mechanisms of the Epilepsies

Jasper's Basic Mechanisms of the Epilepsies [Internet]. 4th edition.

Show details

Embryonic Stem Cell Therapy for Intractable Epilepsy

, , and .

Author Information

Stem cell therapies for treating or curing severe neurological disorders and intractable epilepsy are envisioned for regenerative medicine and neurology of the future. Studies of how the human nervous system develops from stem cells are yielding clues for how to direct pluripotent stem cells into specific neural and glial fates. Reagents and protocols to grow human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in xenofree conditions are now available. Transgenic technology has been developed using Bacterial Artificial Chromosomes (BACs) to force transcription factor expression in these cell lines, making it feasible to direct ESCs and iPSCs toward neural fates to derive functionally distinct classes of neurons, oligodendrocytes and astrocytes comparable to the endogenous cells in different regions of the fetal brain and spinal cord. The remarkable progress in this field may lead to powerful new stem cell-based therapies for curing human neurological disorders and epilepsy. We review some of the major advantages of deriving specific cell types with this new technology, applications in basic research, and prospects for treating circuit abnormalities and intractable seizures in patients with epilepsy.

Envisioning a world in which humans are able to regenerate severed limbs, rewire neural pathways, and enhance sensory perception has been a mainstay of science fiction novels and movies for decades. While human limb regeneration is still not possible, remarkable developments in the fields of stem cell biology and neuroscience are leading the way for stem cell-based therapies to amend brain and spinal cord damage and repair sensory organs. In this chapter, we discuss recent efforts to derived neural stem cells from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) and applications to treating conditions such as temporal lobe epilepsy and neurodegenerative diseases.

Information about the spatiotemporal patterns of transcription factor expression in the developing nervous system suggests that the major classes of neurons in the brain and spinal cord become specified by combinatorial codes of transcription factors. Studies aimed at directing pluripotent stem cells towards neural fates have utilized this information to monitor the differentiation of ESCs and iPSCs into neural progenitors. There are several examples where sufficient advances in production of clinical grade cells have led to clinical trial for neurological disorders. Geron initiated the first clinical trial with transplanted human ESC-derived glial progenitors for repairing spinal cord injuries in paraplegic patients (http://www.geron.com/). Due to financial constraints however, the company halted the trial and withdrew from the field of stem cell therapy. A second clinical trial with retinal grafts of hESC-derived retinal pigment epithelial cells will establish whether blindness caused by macular degeneration is treatable by this approach. This clinical trial, recently carried out by Advanced Cell Technologies, Inc. suggested that the hESC derived retinal pigmented epithelial cells showed no signs of hyperproliferation, tumorigenicity, ectopic tissue formation, or tissue rejection after 4 months of cell transplant. In both cases, the cell-based therapies were extensively validated in animal models of the disorders. Systematic studies of the fates and functional properties of neurons and glia derived from ESCs and iPSCs in experimental models of epilepsy are still in their infancy. Relatively few studies have evaluated the efficacy of transplanting different cell types for seizure control in epilepsy.

Might Stem Cell Therapies be Effective for Controlling Seizures in Epilepsy?

Spontaneous seizures in patients may induce changes in gene expression and trigger widespread inflammation. It is not well understood how these and other seizure-induced changes in the brain will influence the survival, differentiation, or integration of transplanted neural precursors and therefore, stem cell cures for complex and heterogeneous neurological disorders such as temporal lobe epilepsy may be many years in the future.

Mesial temporal lobe epilepsy (MTLE) is often acquired after prolonged status epilepticus caused by prolonged high fever, a brain tumor, stroke, or traumatic brain injury, but is also found in patients diagnosed with Alzheimer’s disease. Following the initial brain injury, neural plasticity is thought to lead to an imbalance between synaptic excitation and inhibition in the dentate gyrus, abetting epileptogenesis.1 Magnetic resonance imaging studies show that hippocampal atrophy is common in patients with childhood-onset MTLE 2, and seizures in childhood are associated with cognitive decline.3, 4 Partial complex epilepsy involving the mesial temporal lobes can be difficult to control with anti-convulsant medications, and the high doses that are often required can cause debilitating cognitive side effects and toxicity. Surgical resection of hippocampus or severing the corpus callosum are alternative therapeutic interventions that may be beneficial for eliminating the seizure focus or reducing the spread of seizures,5 but hippocampal resection can only be used in MTLE patients with a well-defined and unilateral seizure focus. When pharmacological and surgical approaches are not feasible, stem cell therapies might be an alternative therapy and it also offers the additional potential for cell replacement and neural circuit repair. The availability of human embryonic stem cells (hESCs) and strategies for directing their differentiation toward specific types of forebrain neurons and glia is beginning to offer hope that a stem cell cure may be on the horizon for treating hippocampal sclerosis in some forms of acquired epilepsy.6,7

Repairing Dysfunctional Neural Circuitry in Temporal Lobe Epilepsy

Epileptogenesis refers to cellular and molecular changes occurring during a latent period after an initial insult or seizure, when the brain rewires and becomes prone to spontaneous seizures. Epileptogenesis is hypothesized to be caused by disruption of the normal balance between inhibitory and excitatory connections within limbic circuits.8 One hypothetical mechanism in epileptogenesis is the loss of GABAergic interneurons and inhibitory synapses with granule cells after an initial precipitating injury. GABA is the principle inhibitory neurotransmitter in the adult neocortex and hippocampus, where it constrains the spread of neuronal excitation. The GABA producing interneurons also modulate and integrate information in the cortex and hippocampus, by synchronizing cortical oscillations underlying brain function and preventing development of hyperexcitability and epileptiform activity. Two of the hallmark neuropathological changes in patients with MTLE resulting from traumatic brain injury or prolonged febrile seizures, are reduced numbers of hilar interneurons and mossy cells in the dentate gyrus.2,9,10

In experimental MTLE in adult rodents, prolonged seizures or head trauma lead to hyperexcitability of granule neurons11 and loss of functional subclasses of GABAergic interneurons that co-express neuropeptide Y or somatostatin.12–14 These observations have spurred efforts to repair the damaged neural circuits with GABAergic neuron precursors generated from embryonic stem cells or fetal neural progenitor cells, as discussed below.

In both the neocortex and hippocampus, inhibitory cells also regulate networks of synaptically interconnected excitatory pyramidal neurons. Due to recurrent networks of excitatory connections, “runaway excitation” and prolonged burst firing of pyramidal neurons can result when synaptic inhibition is reduced. It has been proposed that prolonged status epilepticus impairs the efficacy of inhibitory GABAergic synaptic transmission onto granule neurons,15, caused by dysregulation of GABAA receptors.16, 17 In addition, recent studies in the pilocarpine model suggest that despite a significant loss of hilar GABAergic interneurons, the residual hippocampal interneurons undergo massive sprouting, resulting in a net increase in GABAergic synapses in the inner molecular layer of the dentate gyrus.18 Failure of these new connections to regulate dentate granule neuron hyperexcitability further supports the hypothesis that inhibitory synaptic transmission becomes dysfunctional in some forms of acquired MTLE.18,19 Developmental disorders affecting the specification and migration of GABAergic interneurons in neocortical and hippocampal regions have also been shown to cause abnormal neuronal firing properties and spontaneous seizures.20–24

Taken together, the evidence from human neuroimaging data, postmortem histological studies of tissue from MTLE patients, and experimental models in rodents supports the idea that defective inhibitory neurotransmission in the hippocampus is a key factor in epileptogenesis and recurrent spontaneous seizures. As the above examples show however, effective cell replacement in acquired forms of MTLE is likely to require extensive integration of specific functional types of GABAergic neurons into the dentate gyrus and correction of some of the other deficits in limbic circuits that underlie epileptogenesis.

In addition to requiring selective cell replacement in MTLE, effective therapies may need to address activity-dependent changes in gene expression induced by seizure activity that alter patterns of granule cell neurogenesis in the dentate gyrus.25 Spontaneous recurrent seizures in MTLE are also associated with sprouting of mossy fibers from granule neurons, creating new excitatory synaptic connections in the inner molecular layer of the dentate gyrus.26–28 While the exact mechanisms remain unclear, aberrant migration of the new neurons and abnormal axonal and dendritic growth of granule neurons contribute to network dysfunction in MTLE.25, 29–31

Glial cell involvement in chronic epilepsy is an additional feature adding to the complexity of the cellular and molecular changes. Glia cells are an important source of the anticonvulsant molecule adenosine and augmenting adenosine levels has a powerful anticonvulsant effect.32–35 However, glial cells also produce proinflammatory molecules that contribute to hyperexcitability. Taken together these studies implicate multiple cell types and pathophysiological processes in acquired focal temporal lobe epilepsy. Even with the advent of new methods for replacing specific types of neurons and glia in the brain by stem cell therapy, treatment strategies may need to be devised that incorporate multiple approaches to correct the defects, including gene therapy, neuroprotection, and dietary modifications such as the ketogenic diet.

Stem Cells for Neurodegeneration and Epilepsy

Fetal neural precursors (NPCs), adult neural stem cells, mesenchymal stem cells (MSCs), cord blood cells, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs) are all being tested for therapeutic effects in experimental models of neurodegenerative diseases and epilepsy. By definition, neural stem cells are self-renewing and can generate neurons or glial cells through asymmetric cell division.

Recent studies demonstrated that stem cells not only exist in the developing embryo, but also in the adult body and brain. The discovery and isolation of neural stem cells from the fetal and adult nervous system has shown that they retain the potential to generate the three major cell types in the central nervous system, namely neurons, astrocytes and oligodendrocytes. Once molecules that govern stem cell self-renewal were identified, it became possible to harvest neural stem cells from the fetal or adult nervous system, expand populations as neurospheres in vitro, or transplant them directly after removal from fetal brains. To date, significantly more progress has been made examining the efficacy of grafts derived from fetal progenitors or genetically modified cell lines, compared to neural stem cells derived from ESCs or iPSCs, for suppressing seizures in experimental models of TLE, ion channel mutations, or developmental disorders of interneurons associated with spontaneous seizures.36–41

Classification systems for the different brain-specific stem cells are still evolving.42 Populations of neural stem cells in the adult nervous system reside within a specialized stem cell niche surrounding the lateral ventricles, called the subependymal zone. Another specialized niche is the subgranular zone of the dentate gyrus, but here, the stem cells are defined as progenitors, because separate populations appear to give rise to neurons or glial cells and have very limited self-renewal capacity.

In tissues of the adult body, stem cells are hard to find, but they do exist. An advantage of patient-derived stem cells is that they may be used for autologous transplants, avoiding problems with immune incompatibility and rejection.43,44 Increasingly neural stem cells derived from patients with genetic disorders of the nervous system are being studied in vitro to gain insights into basic disease mechanisms and for drug discovery. However, limited availability of human fetal tissue precludes widespread use of fetal neural stem cells for most clinical or biotechnology applications. To circumvent this problem, protocols have been developed to improve the yield of fetal or adult neural stem cells from the brain by propagating them as neurosphere-forming cells. While rarely tumorigenic when transplanted into the brain, propagation of hNPCs in culture may select for chromosomal aneuploidy, therefore cytogenetic screening of hNPCs is necessary prior to their use in clinical applications.45

Pluripotent Stem Cells

ESCs are pluripotent stem cells derived from the inner cell mass of human or mouse blastocyst. They have the capacity to generate all tissue types of the embryo. Protocols for differentiating ESCs into neural stem cells in vitro have been developed for adherent ESC lines as well as those that require an initial stage of growth as embryoid bodies and they were shown to undergo extensive migration and integration after transplantation into the developing rodent brain.46–48 The three main in vitro approaches for generating neural stem cells (NSCs) from ESCS include the formation of embryoid bodies, growth of neural stem cells in monolayer cultures on feeder layers in the presence o f growth factors such as fibroblast growth factor-2 (FGF-2), or neurosphere cultures grown in the presence of defined growth factors. The challenge has been to obtain neural stem cells that can be patterned with regional identities upon differentiation into specific neural and glial types. Rapid progress toward this goal has been aided by identification of the transcriptional codes that specify functionally distinct subsets of neurons at different levels of the developing nervous system.

Recent modifications to culture conditions resulted in growth of mouse and human ESCs-derived neural stem cells into neural tube-like rosette structures.49 As these neural rosettes grow, they recapitulate some notable features of the growing neural tube. Symmetrical mitotic divisions characterize the early-stage rosettes, without forming postmitotic neurons. There is a close temporal link between the neural rosette stage and its potential for neural patterning, similar to the neural tube. As neural rosettes develop into late-stage rosettes, they divide asymmetrically, producing neurons and glia that migrate away and differentiate. At this stage, radial glial-like progenitors appear in the rosettes and show interkinetic nuclear migration, similar to radial glial cells of the neural tube. They also have apical end feet that form a structure similar to the center of the neural tube. Human ESC-derive neural stem cells also develop laminated structures with distinctive cell populations in the different layers, reminiscent of the cerebral cortex. As shown in Figure 1, human H9 pluripotent stem cells can be differentiated into neural stem cells in adherent cultures. These cells are beyond the neural rosette stage and can be readily patterned toward a wide range of neural and glial fates. Studies have also shown that transplanted ESC-derived human or mouse neural progenitors migrate in the rodent brain after transplantation into the cerebral cortex or hippocampus, and differentiate in a region-specific manner.41,46,50 It is not known whether these cells can provide long-term seizure suppression or neuroprotection in temporal lobe epilepsy.

Figure 1. Neural Induction of hESC to NSCs Followed By Differentiation Into Neuronal and Glial Lineages.

Figure 1

Neural Induction of hESC to NSCs Followed By Differentiation Into Neuronal and Glial Lineages. Human Embryonic Stem Cells (H9) are pluripotent and able to differentiate into all derivatives of the three primary germ layers: ectoderm, endoderm, and mesoderm.Pluripotency (more...)

Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) are genetically reprogrammed adult cells which exhibit a pluripotent stem cell like state like that of human embryonic stem cells. IPSCs may be derived by several different methods that induce select gene expression to confer pluripotency.51–53 Functionally distinct types of neurons have been generated from iPSCs and studies are now examining their ability to migrate and successfully modify symptoms of neurological disease.54–56 Some of the advantages of studying iPSC-derived neurons from patients with epilepsy include the possibility of gaining insights at the cellular level into how a particular genetic mutation affects neuronal excitability, migration, dendritic or axonal differentiation, or synapse formation. This approach is now being used to study iPSCs from patients with inherited forms of epilepsy. These cells may also be quite useful for screening anti-convulsant drugs. Based on their cellular responses to different drug compounds, more effective anti-convulsant drugs can be selected and then administered to individual patients.

Directed Differentiation of ESCS and IPSCS

Recent advances in directing the fates of hESCs and iPSCs toward neural cell types that have relevance to temporal lobe epilepsy have utilized defined media conditions, reporter genes, or bacterial artificial chromosomes to select for neural stem cells with GABAergic fates.50,57 For example transcriptional codes for specifying GABAergic interneuron subtypes have been identified58,59 and this information has been used to direct ESC-derived neural progenitors to differentiate into GABAergic interneurons.60 In one study, floating stem cell aggregates were first generated to obtain neural progenitors with a transgene, Lhx6-GFP. These were then grown on adherent substrates in the presence of growth factors and the signaling molecule sonic hedgehog, to obtain ventral telencephalon neural progenitors. By harvesting the neural precursors at this stage, they were able to use the fluorescent transgene in fluorescence activated cell sorting (FACS) to identify and prospectively isolate the Lhx6-expressing GABAergic progenitors prior to transplantation.60 When the isolated interneuron precursors were then transplanted into the developing mouse brain, they differentiated into functionally defined types of interneurons and exhibited mature electrophysiological properties. Studies are now beginning to systematically test whether transplants of interneuron progenitors obtained with similar protocols, have disease-modifying effects in experimental models of temporal lobe epilepsy.

Bacterial Artificial Chromosome Transgenesis

Another powerful approach is to force the differentiation of hESCs derived neural stem cells toward particular neural fates by introducing Bacterial Artificial Chromosomes (BAC) into the cells. This approach, called BAC transgenesis, is a novel tool to define and isolate different neural and glial lineages from ES cells.61–63 This approach has used the GENSAT library of BACs, which have been engineered to express GFP under the control of key genes related to neural development. To obtain stable BAC transgenesis in hESCs, the cells had to be dissociated into single cell suspensions using enzymatic digestion and an inhibitor of the Rho-associated kinase. This relatively simple step provided a substantial improvement in survival and cloning efficiency. The BACS were then nucleofected into the dissociated hESCs, and selected for antibiotic resistance to obtain hESCs with different GFP transgenes that report differentiation into motor neuron or dopamine neuron fates.

Clinical Grade Pluripotent Stem Cells In Xeno and Feeder-Free Culture Systems

Successful stem cell therapies for CNS diseases require large-scale production of neural stem cells from hESC or iPSCs. In addition, such neural stem cells should be nontransformed and well characterized, with adequate bioprocess control such as safety, sterility, traceability. These cells upon transplantation should differentiate into appropriate target cell type, survive in diseased CNS tissue and should integrate with the existing neural network to improve the functional efficacy and clinical outcome.

A variety of protocols and cell culture conditions have been reported for the generation, expansion and differentiation of NSC from hESCs. A typical induction protocol from hESCs to hNSCs is shown in a schematic diagram (Figure 1) and this can be achieved either by spontaneous or directed differentiation methods.64–66 Some of the earliest protocols used serum free culture conditions with either N2 or B27 neural supplements, but the process was not robust enough for reproducibility and scalability and neural induction efficiency was low (0.2% hESC to hNPC) with spontaneous differentiation.67 But higher efficiency for neural induction was achieved by adding retinoic acid,68 medium conditioned with stromal cells,69 or BMP pathway inhibitors.70

For human cell therapy however, using animal serum in the media, stromal feeder cells derived from animals, co-cultures, or pre-conditioned media was incompatible because animal cell- and serum-free conditions are necessary. Guidelines for the manufacture of biologics have been developed and codified under CMC (chemistry and manufacturing controls) and GMP (good manufacturing practices). Deriving the first hESC line with these properties for clinical applications was an important advance.71 It was necessary to demonstrate the ability to obtain pure populations of long-term self-renewing rosette-type hESC-derived neural stem cells that exhibited extensive self-renewal, clonogenicity, and stable neurogenesis upon transplantation.72 Recently a hESC line was generated that has the capacity to produce dopaminergic precursors in completely xenofree cultures using four-steps for producing functional dopaminergic neurons for clinical purposes.73 Neurons generated by this process were shown to survive in experimental animal models of Parkinson’s disease. A third important advance was development of an efficient method to generate hNSCs from hESC or induced pluripotent stem cells by a combined blockade of SMAD signaling with Noggin and a small molecule, SB431542 to achieve full neural conversion. This method is regulatory complaint, as it obviates the need for intermediary steps of generating embryoid bodies, the use of stromal feeders and even isolation of rosette structures.74 The commercial availability of expandable populations of NSC with stable phenotype marker expression will facilitate efforts to produce NSC under xenofree culture conditions for epilepsy research and clinical applications.

Transplant Studies of Fetal NSC for Seizure Suppression in Experimental Models

Neural stem cells and fetal neural precursors have been transplanted into a variety of different experimental models of neurological disorders including ischemic stroke, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, spinal cord injury, and epilepsy.40, 41, 50, 75–83

Evidence from experimental studies in several models of temporal lobe epilepsy in rodents has provided “proof of concept” that fetal neural stem cell transplants or cell lines genetically engineered to release GABA at multiple sites in the brain reduce seizure severity and/or frequency.36, 84–92 Studies with fetal cell transplants reduce abnormal electrical discharges in the hippocampus or substantia nigra. Moreover, when GABAergic neural precursors from the embryonic medial ganglionic eminence were transplanted into forebrains of young mice, they showed widespread migration across the hippocampus and neocortex. The grafted cells differentiated into distinct types of GABAergic interneurons, functionally integrated, and increased inhibitory tone in the host cortex and hippocampus. 37 These studies serve as proof-of-concept that increasing the number of forebrain GABAergic neurons in several different models of epilepsy can not only raise seizure thresholds in genetic models but also suppress recurrent spontaneous seizures in acquired MTLE. Studies employing fetal neural stem cells are described in detail in additional chapters in this volume (Baraban and Anderson; Shetty).

Challenges in Cell Therapy Approaches to Treat Epilepsy

While there are several advantages of stem cell based therapies over gene therapy and other traditional approaches for treating epilepsy, significant technical hurdles still impede cell therapy for treating epilepsy. Unlike other tissues of the body, the nervous system has a limited capacity for self-repair because mature neurons cannot regenerate, and despite the presence of neural stem cells in the adult brain, their ability to respond to injury is limited. Improving the efficacy of stem cell therapies for replacing neurons or glial cell types destroyed by damage or disease is an extremely active area of investigation. To be successful, grafts of stem cells and their differentiated derivatives in the epileptic brain must not only survive for long periods of time, they also need to migrate correctly to the appropriate sites, integrate, and establish the correct types of synaptic connections with the host brain. The importance of this last point is underscored by studies showing that seizures induce the genesis of ectopically positioned neurons from endogenous neural stem populations, and these ectopic neurons can contribute to increased excitability and epileptogenesis.29,93

Immune incompatibility between the donor and host is one of the more formidable problems in the field of cell replacement therapy. Currently, cell therapies based on fetal cell transplants require that patients receiving fetal stem cells also take immunosuppressive drugs to prevent rejection of fetal cell grafts.40 Autologous stem cell grafts, in which the patient is also the stem cell donor, may help overcome the problem of graft rejection. Another major hurdle for ES based therapy is that the risk of tumor formation is high because they are pluripotent and mitotically active. To address this problem, human ES cells have been engineered with suicide genes to allow elimination if the transplanted cells proliferate excessively or form tumors.94

CONCLUSIONS AND THE PATH FORWARD

Testing the feasibility of ESC-derived neurons for seizure suppression and hippocampal sclerosis in epilepsy will require a better understanding of how seizures alter the environment of the brain. Activity-dependent changes in the expression of a large number of genes have been found in experimental models of epilepsy. Additionally, even brief seizures may cause epigenetic changes that alter gene expression. Evidence for host brain influences on transplanted neural stem cells suggests that these local changes in the brain’s milieu may be powerful influences on stem cell survival and migration after transplantation.

Additional issues that need to be surmounted for further advances in stem cell therapies for epilepsy include a better ability to track the transplanted cells after transplantation in the human brain. It will also be necessary to achieve long-term survival of transplants, circumvent the immunological problem of graft rejection, and tailor therapies for individual patients. The development of protocols for directing iPS cells into particular cell fates is one way to remove immunological barriers, since these cells can be generated from the patient’s skin and offer the possibility of transplanting back into the same patient. Lastly, before moving into the clinic, it will be necessary to produce “clinical-grade” human pluripotent cell -derived neural stem cells that differentiate into particular classes of GABAergic interneurons or other cell types that are injured in epilepsy.

With further new technological advances in the field of stem cell biology, cell therapies to treat neurological disorders such as epilepsy may soon become feasible. Stem cell transplants that employ neural precursors for subclasses of GABAergic interneurons show great promise for controlling “run-away” excitation in the brain and spontaneous seizures in acquired forms of temporal lobe epilepsy. However, further studies are needed in a range of translational models of temporal lobe epilepsy to determine whether hESC- or iPSC-derived neurons transplanted into the hippocampus have the capacity to survive over the long term, form synapses with their appropriate synaptic targets, and regulate pyramidal and granule neuron hyper-excitability, without causing adverse neurological side-effects.

ACKNOWLEDGEMENTS

The authors thank Drs. Soojung Shin and Yiping Yan for images of neural induction from hESC and Dr. Paul Lombroso and Xu Maisano for comments on the manuscript.

REFERENCES

1.
Chang BS, Lowenstein DH. Epilepsy N Engl J Med. 2003;349:1257–66. [PubMed: 14507951]
2.
Mathern GW, Pretorius JK, Babb TL. Influence of the type of initial precipitating injury and at what age it occurs on course and outcome in patients with temporal lobe seizures. J Neurosurg. 1995;82:220–7. [PubMed: 7815149]
3.
Bjornaes H, Stabell K, Henriksen O, Loyning Y. The effects of refractory epilepsy on intellectual functioning in children and adults. A longitudinal study. Seizure. 2001;10:250–9. [PubMed: 11466020]
4.
Kolk A, Talvik T. Cognitive outcome of children with early-onset hemiparesis. J Child Neurol. 2000;15:581–7. [PubMed: 11019788]
5.
Engel J Jr, Wiebe S, French J, Sperling M, Williamson P, Spencer D, Gumnit R, Zahn C, Westbrook E, Enos B. Practice parameter: temporal lobe and localized neocortical resections for epilepsy: report of the Quality Standards Subcommittee of the American Academy of Neurology in association with the American Epilepsy Society and the American Association of Neurological Surgeons. Neurology. 2003;60:538–47. [PubMed: 12601090]
6.
Naegele JR, Maisano X. Gene and Stem Cell Therapies for Treating Epilepsy. In: Rho JM, Sankar R, Stafstrom CE, editors. Epilepsy: Mechanisms, Models, and Translational Perspectives. Dekker M, Inc. (CRC Press); 2010. pp. 583–601.
7.
Naegele JR, Maisano X, Yang J, Royston S, Ribeiro E. Recent advancements in stem cell and gene therapies for neurological disorders and intractable epilepsy. Neuropharmacology. 2010;58:855–64. [PMC free article: PMC2838966] [PubMed: 20146928]
8.
Cossart R, Bernard C, Ben-Ari Y. Multiple facets of GABAergic neurons and synapses: multiple fates of GABA signalling in epilepsies. Trends Neurosci. 2005;28:108–15. [PubMed: 15667934]
9.
Swartz BE, Houser CR, Tomiyasu U, Walsh GO, DeSalles A, Rich JR, Delgado-Escueta A. Hippocampal cell loss in posttraumatic human epilepsy. Epilepsia. 2006;47:1373–82. [PubMed: 16922884]
10.
de Lanerolle NC, Kim JH, Robbins RJ, Spencer DD. Hippocampal interneuron loss and plasticity in human temporal lobe epilepsy. Brain Res. 1989;495:387–95. [PubMed: 2569920]
11.
Golarai G, Greenwood AC, Feeney DM, Connor JA. Physiological and structural evidence for hippocampal involvement in persistent seizure susceptibility after traumatic brain injury. J Neurosci. 2001;21:8523–37. [PubMed: 11606641]
12.
McDonald AJ, Mascagni F. Immunohistochemical characterization of somatostatin containing interneurons in the rat basolateral amygdala. Brain Res. 2002;943:237–44. [PubMed: 12101046]
13.
Tuunanen J, Halonen T, Pitkanen A. Decrease in somatostatin-immunoreactive neurons in the rat amygdaloid complex in a kindling model of temporal lobe epilepsy. Epilepsy Res. 1997;26:315–27. [PubMed: 9095393]
14.
Choi YS, Lin SL, Lee B, Kurup P, Cho HY, Naegele JR, Lombroso PJ, Obrietan K. Status epilepticus-induced somatostatinergic hilar interneuron degeneration is regulated by striatal enriched protein tyrosine phosphatase. J Neurosci. 2007;27:2999–3009. [PMC free article: PMC2701360] [PubMed: 17360923]
15.
Bonislawski DP, Schwarzbach EP, Cohen AS. Brain injury impairs dentate gyrus inhibitory efficacy. Neurobiol Dis. 2007;25:163–9. [PMC free article: PMC1713625] [PubMed: 17045484]
16.
Coulter DA, Carlson GC. Functional regulation of the dentate gyrus by GABA-mediated inhibition. Prog Brain Res. 2007;163:235–43. [PubMed: 17765722]
17.
Brooks-Kayal AR, Shumate MD, Jin H, Rikhter TY, Coulter DA. Selective changes in single cell GABA(A) receptor subunit expression and function in temporal lobe epilepsy. Nat Med. 1998;4:1166–72. [PubMed: 9771750]
18.
Zhang W, Yamawaki R, Wen X, Uhl J, Diaz J, Prince DA, Buckmaster PS. Surviving hilar somatostatin interneurons enlarge, sprout axons, and form new synapses with granule cells in a mouse model of temporal lobe epilepsy. J Neurosci. 2009;29:14247–56. [PMC free article: PMC2802278] [PubMed: 19906972]
19.
Kobayashi M, Buckmaster PS. Reduced inhibition of dentate granule cells in a model of temporal lobe epilepsy. J Neurosci. 2003;23:2440–52. [PubMed: 12657704]
20.
Cobos I, Broccoli V, Rubenstein JL. The vertebrate ortholog of Aristaless is regulated by Dlx genes in the developing forebrain. J Comp Neurol. 2005;483:292–303. [PubMed: 15682394]
21.
Price MG, Yoo JW, Burgess DL, Deng F, Hrachovy RA, Frost JD Jr, Noebels JL. A triplet repeat expansion genetic mouse model of infantile spasms syndrome, Arx(GCG)10+7, with interneuronopathy, spasms in infancy, persistent seizures, and adult cognitive and behavioral impairment. J Neurosci. 2009;29:8752–63. [PMC free article: PMC2782569] [PubMed: 19587282]
22.
Marsh E, Fulp C, Gomez E, Nasrallah I, Minarcik J, Sudi J, Christian SL, Mancini G, Labosky P, Dobyns W, Brooks-Kayal A, Golden JA. Targeted loss of Arx results in a developmental epilepsy mouse model and recapitulates the human phenotype in heterozygous females. Brain. 2009;132(Pt 6):1563–76. [PMC free article: PMC2685924] [PubMed: 19439424]
23.
Martins GJ, Plachez C, Powell EM. Loss of embryonic MET signaling alters profiles of hippocampal interneurons. Dev Neurosci. 2007;29:143–58. [PubMed: 17148957]
24.
Powell EM, Campbell DB, Stanwood GD, Davis C, Noebels JL, Levitt P. Genetic disruption of cortical interneuron development causes region- and GABA cell type-specific deficits, epilepsy, and behavioral dysfunction. J Neurosci. 2003;23:622–31. [PubMed: 12533622]
25.
Ma DK, Jang MH, Guo JU, Kitabatake Y, Chang ML, Pow-Anpongkul N, Flavell RA, Lu B, Ming GL, Song H. Neuronal activity-induced Gadd45b promotes epigenetic DNA demethylation and adult neurogenesis. Science. 2009;323:1074–7. [PMC free article: PMC2726986] [PubMed: 19119186]
26.
Tauck DL, Nadler JV. Evidence of functional mossy fiber sprouting in hippocampal formation of kainic acid-treated rats. J Neurosci. 1985;5:1016–22. [PubMed: 3981241]
27.
Sloviter RS, Zappone CA, Harvey BD, Frotscher M. Kainic acid-induced recurrent mossy fiber innervation of dentate gyrus inhibitory interneurons: possible anatomical substrate of granule cell hyper-inhibition in chronically epileptic rats. J Comp Neurol. 2006;494:944–60. [PMC free article: PMC2597112] [PubMed: 16385488]
28.
Shibley H, Smith BN. Pilocarpine-induced status epilepticus results in mossy fiber sprouting and spontaneous seizures in C57BL/6 and CD-1 mice. Epilepsy Res. 2002;49:109–20. [PubMed: 12049799]
29.
Parent JM. Adult neurogenesis in the intact and epileptic dentate gyrus. Prog Brain Res. 2007;163:529–40. [PubMed: 17765736]
30.
Scharfman HE, Gray WP. Relevance of seizure-induced neurogenesis in animal models of epilepsy to the etiology of temporal lobe epilepsy. Epilepsia. 2007;48(Suppl 2):33–41. [PMC free article: PMC2504501] [PubMed: 17571351]
31.
Ribak CE, Tran PH, Spigelman I, Okazaki MM, Nadler JV. Status epilepticus-induced hilar basal dendrites on rodent granule cells contribute to recurrent excitatory circuitry. J Comp Neurol. 2000;428:240–53. [PubMed: 11064364]
32.
Boison D. Adenosine kinase, epilepsy and stroke: mechanisms and therapies. Trends Pharmacol Sci. 2006;27:652–8. [PubMed: 17056128]
33.
Boison D. The adenosine kinase hypothesis of epileptogenesis. Prog Neurobiol. 2008;84:249–62. [PMC free article: PMC2278041] [PubMed: 18249058]
34.
Boison D. Adenosine augmentation therapies (AATs) for epilepsy: prospect of cell and gene therapies. Epilepsy Res. 2009;85:131–41. [PMC free article: PMC2713801] [PubMed: 19428218]
35.
Masino SA, Kawamura M, Wasser CA, Pomeroy LT, Ruskin DN. Adenosine, ketogenic diet and epilepsy: the emerging therapeutic relationship between metabolism and brain activity. Curr Neuropharmacol. 2009;7:257–68. [PMC free article: PMC2769009] [PubMed: 20190967]
36.
Castillo CG, Mendoza S, Freed WJ, Giordano M. Intranigral transplants of immortalized GABAergic cells decrease the expression of kainic acid-induced seizures in the rat. Behav Brain Res. 2006;171:109–15. [PubMed: 16677720]
37.
Alvarez-Dolado M, Calcagnotto ME, Karkar KM, Southwell DG, Jones-Davis DM, Estrada RC, Rubenstein JL, Alvarez-Buylla A, Baraban SC. Cortical inhibition modified by embryonic neural precursors grafted into the postnatal brain. J Neurosci. 2006;26:7380–9. [PMC free article: PMC1550786] [PubMed: 16837585]
38.
Baraban SC, Southwell DG, Estrada RC, Jones DL, Sebe JY, Alfaro-Cervello C, Garcia-Verdugo JM, Rubenstein JL, Alvarez-Buylla A. Reduction of seizures by transplantation of cortical GABAergic interneuron precursors into Kv1.1 mutant mice. Proc Natl Acad Sci USA. 2009;106:15472–7. [PMC free article: PMC2741275] [PubMed: 19706400]
39.
Bengzon J, Kokaia Z, Lindvall O. Specific functions of grafted locus coeruleus neurons in the kindling model of epilepsy. Exp Neurol. 1993;122:143–54. [PubMed: 8339784]
40.
Bjorklund A, Lindvall O. Cell replacement therapies for central nervous system disorders. Nat Neurosci. 2000;3:537–44. [PubMed: 10816308]
41.
Carpentino JE, Hartman NW, Grabel LB, Naegele JR. Region-specific differentiation of embryonic stem cell-derived neural progenitor transplants into the adult mouse hippocampus following seizures. J Neurosci Res. 2008;86:512–24. [PubMed: 17918739]
42.
Seaberg RM, van der Kooy D. Stem and progenitor cells: the premature desertion of rigorous definitions. Trends Neurosci. 2003;26:125–31. [PubMed: 12591214]
43.
Gage FH. Mammalian neural stem cells. Science. 2000;287:1433–8. [PubMed: 10688783]
44.
Kokovay E, Shen Q, Temple S. The incredible elastic brain: how neural stem cells expand our minds. Neuron. 2008;60:420–9. [PubMed: 18995816]
45.
Sareen D, McMillan E, Ebert AD, Shelley BC, Johnson JA, Meisner LF, Svendsen CN. Chromosome 7 and 19 trisomy in cultured human neural progenitor cells. PLoS One. 2009;4(10):e7630. [PMC free article: PMC2765070] [PubMed: 19898616]
46.
Tabar V, Panagiotakos G, Greenberg ED, Chan BK, Sadelain M, Gutin PH, Studer L. Migration and differentiation of neural precursors derived from human embryonic stem cells in the rat brain. Nat Biotechnol. 2005;23:601–6. [PubMed: 15852001]
47.
Zhang SC, Wernig M, Duncan ID, Brustle O, Thomson JA. In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat Biotechnol. 2001;19:1129–33. [PubMed: 11731781]
48.
Reubinoff BE, Itsykson P, Turetsky T, Pera MF, Reinhartz E, Itzik A, Ben-Hur T. Neural progenitors from human embryonic stem cells. Nat Biotechnol. 2001;19:1134–40. [PubMed: 11731782]
49.
Elkabetz Y, Panagiotakos G, Al Shamy G, Socci ND, Tabar V, Studer L. Human ES cell-derived neural rosettes reveal a functionally distinct early neural stem cell stage. Genes Dev. 2008;22:152–65. [PMC free article: PMC2192751] [PubMed: 18198334]
50.
Maisano X, Carpentino J, Becker S, Lanza R, Aaron G, Grabel L, Naegele JR. Embryonic stem cell-derived neural precursor grafts for treatment of temporal lobe epilepsy. Neurotherapeutics. 2009;6:263–77. [PMC free article: PMC2830617] [PubMed: 19332319]
51.
Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131:861–72. [PubMed: 18035408]
52.
Takahashi K, Okita K, Nakagawa M, Yamanaka S. Induction of pluripotent stem cells from fibroblast cultures. Nat Protoc. 2007;2:3081–9. [PubMed: 18079707]
53.
Kim SU, de Vellis J. Stem cell-based cell therapy in neurological diseases: a review. J Neurosci Res. 2009;87:2183–200. [PubMed: 19301431]
54.
Karumbayaram S, Novitch BG, Patterson M, Umbach JA, Richter L, Lindgren A, Conway AE, Clark AT, Goldman SA, Plath K, Wiedau-Pazos M, Kornblum HI, Lowry WE. Directed differentiation of human-induced pluripotent stem cells generates active motor neurons. Stem Cells. 2009;27:806–11. [PMC free article: PMC2895909] [PubMed: 19350680]
55.
Dimos JT, Rodolfa KT, Niakan KK, Weisenthal LM, Mitsumoto H, Chung W, Croft GF, Saphier G, Leibel R, Goland R, Wichterle H, Henderson CE, Eggan K. Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science. 2008;321:1218–21. [PubMed: 18669821]
56.
Wernig M, Zhao JP, Pruszak J, Hedlund E, Fu D, Soldner F, Broccoli V, Constantine-Paton M, Isacson O, Jaenisch R. Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson’s disease. Proc Natl Acad Sci USA. 2008;105:5856–61. [PMC free article: PMC2311361] [PubMed: 18391196]
57.
Cai C, Grabel L. Directing the differentiation of embryonic stem cells to neural stem cells. Dev Dyn. 2007;236:3255–66. [PubMed: 17823944]
58.
Butt SJ, Cobos I, Golden J, Kessaris N, Pachnis V, Anderson S. Transcriptional regulation of cortical interneuron development. J Neurosci. 2007;27:11847–50. [PubMed: 17978022]
59.
Butt SJ, Fuccillo M, Nery S, Noctor S, Kriegstein A, Corbin JG, Fishell G. The temporal and spatial origins of cortical interneurons predict their physiological subtype. Neuron. 2005;48:591–604. [PubMed: 16301176]
60.
Maroof AM, Brown K, Shi SH, Studer L, Anderson SA. Prospective isolation of cortical interneuron precursors from mouse embryonic stem cells. J Neurosci. 2010;30:4667–75. [PMC free article: PMC2868507] [PubMed: 20357117]
61.
Placantonakis DG, Tomishima MJ, Lafaille F, Desbordes SC, Jia F, Socci ND, Niale A, Lee H, Harrison N, Studer L, Tabar VS. Enriched motor neuron populations derived from bacterial artificial chromosome-transgenic human embryonic stem cells. Clin Neurosurg. 2009;56:125–32. [PubMed: 20214043]
62.
Placantonakis DG, Tomishima MJ, Lafaille F, Desbordes SC, Jia F, Socci ND, Viale A, Lee H, Harrison N, Tabar V, Studer L. BAC transgenesis in human embryonic stem cells as a novel tool to define the human neural lineage. Stem Cells. 2009;27:521–32. [PubMed: 19074416]
63.
Tomishima MJ, Hadjantonakis AK, Gong S, Studer L. Production of green fluorescent protein transgenic embryonic stem cells using the GENSAT bacterial artificial chromosome library. Stem Cells. 2007;25:39–45. [PMC free article: PMC2881625] [PubMed: 16990587]
64.
Schulz TC, Palmarini GM, Noggle SA, Weiler DA, Mitalipova MM, Condie BG. Directed neuronal differentiation of human embryonic stem cells. BMC Neurosci. 2003;4:27. [PMC free article: PMC272931] [PubMed: 14572319]
65.
Gerrard L, Rodgers L, Cui W. Differentiation of human embryonic stem cells to neural lineages in adherent culture by blocking bone morphogenetic protein signaling. Stem Cells. 2005;23:1234–41. [PubMed: 16002783]
66.
Shin S, Mitalipova M, Noggle S, Tibbitts D, Venable A, Rao R, Stice SL. Long-term proliferation of human embryonic stem cell-derived neuroepithelial cells using defined adherent culture conditions. Stem Cells. 2006;24:125–38. [PubMed: 16100006]
67.
Tropepe V, Hitoshi S, Sirard C, Mak TW, Rossant J, van der Kooy D. Direct neural fate specification from embryonic stem cells: a primitive mammalian neural stem cell stage acquired through a default mechanism. Neuron. 2001;30:65–78. [PubMed: 11343645]
68.
Eiges R, Schuldiner M, Drukker M, Yanuka O, Itskovitz-Eldor J, Benvenisty N. Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells. Curr Biol. 2001;11:514–8. [PubMed: 11413002]
69.
Perrier AL, Tabar V, Barberi T, Rubio ME, Bruses J, Topf N, Harrison NL, Studer L. Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc Natl Acad Sci U S A. 2004 Aug 24;101(34:):12543–8. [PMC free article: PMC515094] [PubMed: 15310843]
70.
Itsykson P, Ilouz N, Turetsky T, Goldstein RS, Pera MF, Fishbein I, Segal M, Reubinoff BE. Derivation of neural precursors from human embryonic stem cells in the presence of noggin. Mol Cell Neurosci. 2005;30:24–36. [PubMed: 16081300]
71.
Klimanskaya I, Chung Y, Meisner L, Johnson J, West MD, Lanza R. Human embryonic stem cells derived without feeder cells. Lancet. 2005;365(9471):1636–41. [PubMed: 15885296]
72.
Koch P, Opitz T, Steinbeck JA, Ladewig J, Brustle O. A rosette-type, self-renewing human ES cell-derived neural stem cell with potential for in vitro instruction and synaptic integration. Proc Natl Acad Sci USA. 2009;106:3225–30. [PMC free article: PMC2651316] [PubMed: 19218428]
73.
Swistowski A, Peng J, Liu Q, Mali P, Rao MS, Cheng L, Zeng X. Efficient Generation of Functional Dopaminergic Neurons from Human Induced pluripotent Stem Cells under Defined Conditions. Stem Cells. 2010;28:1893–904. [PMC free article: PMC2996088] [PubMed: 20715183]
74.
Chambers SM, Fasano CA, Papapetrou EP, Tomishima M, Sadelain M, Studer L. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol. 2009;27:275–80. [PMC free article: PMC2756723] [PubMed: 19252484]
75.
Lindvall O. Clinical application of neuronal grafts in Parkinson’s disease. J Neurol. 1994;242(1 Suppl 1):S54–6. [PubMed: 7699411]
76.
Zaman V, Shetty AK. Fetal hippocampal CA3 cell grafts transplanted to lesioned CA3 region of the adult hippocampus exhibit long-term survival in a rat model of temporal lobe epilepsy. Neurobiol Dis. 2001;8:942–52. [PubMed: 11741390]
77.
Turner DA, Shetty AK. Clinical prospects for neural grafting therapy for hippocampal lesions and epilepsy. Neurosurgery. 2003;52:632–44. discussion 41–4. [PubMed: 12590689]
78.
Ruschenschmidt C, Koch PG, Brustle O, Beck H. Functional properties of ES cell-derived neurons engrafted into the hippocampus of adult normal and chronically epileptic rats. Epilepsia. 2005;46(Suppl 5):174–83. [PubMed: 15987274]
79.
Rao MS, Hattiangady B, Rai KS, Shetty AK. Strategies for promoting anti-seizure effects of hippocampal fetal cells grafted into the hippocampus of rats exhibiting chronic temporal lobe epilepsy. Neurobiol Dis. 2007;27:117–32. [PMC free article: PMC3612502] [PubMed: 17618126]
80.
Hattiangady B, Rao MS, Shetty AK. Grafting of striatal precursor cells into hippocampus shortly after status epilepticus restrains chronic temporal lobe epilepsy. Exp Neurol. 2008;212:468–81. [PMC free article: PMC2750902] [PubMed: 18579133]
81.
Aubry L, Bugi A, Lefort N, Rousseau F, Peschanski M, Perrier AL. Striatal progenitors derived from human ES cells mature into DARPP32 neurons in vitro and in quinolinic acid-lesioned rats. Proc Natl Acad Sci U S A. 2008;105:16707–12. [PMC free article: PMC2575484] [PubMed: 18922775]
82.
Bacigaluppi M, Pluchino S, Martino G, Kilic E, Hermann DM. Neural stem/precursor cells for the treatment of ischemic stroke. J Neurol Sci. 2008;265:73–7. [PubMed: 17610905]
83.
Raedt R, Van Dycke A, Vonck K, Boon P. Cell therapy in models for temporal lobe epilepsy. Seizure. 2007;16:565–78. [PubMed: 17566770]
84.
Chu K, Kim M, Jung KH, Jeon D, Lee ST, Kim J, Jeong SW, Kim SU, Lee SK, Shin HS, Roh JK. Human neural stem cell transplantation reduces spontaneous recurrent seizures following pilocarpine-induced status epilepticus in adult rats. Brain Res. 2004;1023:213–21. [PubMed: 15374747]
85.
Clough R, Statnick M, Maring-Smith M, Wang C, Eells J, Browning R, Dailey J, Jobe P. Fetal raphe transplants reduce seizure severity in serotonin-depleted GEPRs. Neuroreport. 1996;8:341–6. [PubMed: 9051807]
86.
Gernert M, Thompson KW, Loscher W, Tobin AJ. Genetically engineered GABA-producing cells demonstrate anticonvulsant effects and long-term transgene expression when transplanted into the central piriform cortex of rats. Exp Neurol. 2002;176:183–92. [PubMed: 12093095]
87.
Kokaia M, Aebischer P, Elmer E, Bengzon J, Kalen P, Kokaia Z, Lindvall O. Seizure suppression in kindling epilepsy by intracerebral implants of GABA- but not by noradrenaline-releasing polymer matrices. Exp Brain Res. 1994;100:385–94. [PubMed: 7813677]
88.
Loscher W, Ebert U, Lehmann H, Rosenthal C, Nikkhah G. Seizure suppression in kindling epilepsy by grafts of fetal GABAergic neurons in rat substantia nigra. J Neurosci Res. 1998;51:196–209. [PubMed: 9469573]
89.
Loscher W, Gernert M, Heinemann U. Cell and gene therapies in epilepsy--promising avenues or blind alleys. Trends Neurosci. 2008;31:62–73. [PubMed: 18201772]
90.
Thompson KW. Genetically engineered cells with regulatable GABA production can affect afterdischarges and behavioral seizures after transplantation into the dentate gyrus. Neuroscience. 2005;133:1029–37. [PubMed: 15927406]
91.
Thompson KW, Suchomelova LM. Transplants of cells engineered to produce GABA suppress spontaneous seizures. Epilepsia. 2004;45:4–12. [PubMed: 14692901]
92.
Waldau B, Hattiangady B, Kuruba R, Shetty AK. Medial ganglionic eminence-derived neural stem cell grafts ease spontaneous seizures and restore GDNF expression in a rat model of chronic temporal lobe epilepsy. Stem Cells. 2010;28:1153–64. [PMC free article: PMC2933789] [PubMed: 20506409]
93.
Scharfman HE. Functional implications of seizure-induced neurogenesis. Adv Exp Med Biol. 2004;548:192–212. [PMC free article: PMC1839060] [PubMed: 15250595]
94.
Schuldiner M, Itskovitz-Eldor J, Benvenisty N. Selective ablation of human embryonic stem cells expressing a “suicide” gene. Stem Cells. 2003;21:257–65. [PubMed: 12743320]
Copyright © 2012, Michael A Rogawski, Antonio V Delgado-Escueta, Jeffrey L Noebels, Massimo Avoli and Richard W Olsen.

All Jasper's Basic Mechanisms of the Epilepsies content, except where otherwise noted, is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported license, which permits copying, distribution and transmission of the work, provided the original work is properly cited, not used for commercial purposes, nor is altered or transformed.

Bookshelf ID: NBK98165PMID: 22787645

Views

Related information

  • PMC
    PubMed Central citations
  • PubMed
    Links to PubMed

Similar articles in PubMed

See reviews...See all...

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...