Functional Recovery Following Traumatic Spinal Cord Injury Mediated by a Unique Polymer Scaffold Seeded with Neural Stem Cells

Proceedings of the National Academy of Sciences of the United States of America. Mar, 2002  |  Pubmed ID: 11867737

To better direct repair following spinal cord injury (SCI), we designed an implant modeled after the intact spinal cord consisting of a multicomponent polymer scaffold seeded with neural stem cells. Implantation of the scaffold-neural stem cells unit into an adult rat hemisection model of SCI promoted long-term improvement in function (persistent for 1 year in some animals) relative to a lesion-control group. At 70 days postinjury, animals implanted with scaffold-plus-cells exhibited coordinated, weight-bearing hindlimb stepping. Histology and immunocytochemical analysis suggested that this recovery might be attributable partly to a reduction in tissue loss from secondary injury processes as well as in diminished glial scarring. Tract tracing demonstrated corticospinal tract fibers passing through the injury epicenter to the caudal cord, a phenomenon not present in untreated groups. Together with evidence of enhanced local GAP-43 expression not seen in controls, these findings suggest a possible regeneration component. These results may suggest a new approach to SCI and, more broadly, may serve as a prototype for multidisciplinary strategies against complex neurological problems.

Transplanted Clonal Neural Stem-like Cells Respond to Remote Photic Stimulation Following Incorporation Within the Suprachiasmatic Nucleus

Experimental Neurology. Apr, 2002  |  Pubmed ID: 11922658

Multipotent neural stem-like cells (NSCs) obtained from one brain region and transplanted to another region appear to differentiate into neuronal and glial phenotypes indigenous to the implantation site. Whether these donor-derived cells are appropriately integrated remains unanswered. In order to test this possibility, we exploited the suprachiasmatic nucleus (SCN) of the hypothalamus, site of a known circadian clock, as a novel engraftment target. When a clone of NSCs initially derived from neonatal mouse cerebellum was transplanted into mouse embryos, the cells incorporated within the SCN over a narrow gestational window that corresponded to the conclusion of SCN neurogenesis. Immunocytochemical staining suggested that donor-derived cells in the SCN synthesized a peptide neurotransmitter (arginine vasopressin) characteristic of SCN neurons. Donor-derived SCN cells reacted to light pulses by expressing immunoreactive c-Fos protein in a pattern that is appropriate for native SCN cells. This region-specific and physiologically appropriate response to the natural stimulation of a remote sensory input implies that donor-derived and endogenous cells formed true SCN chimeras, suggesting that exogenous NSCs engrafted to ectopic locations can integrate in a meaningful fashion.

Limitations in Brain Repair

Nature Medicine. Sep, 2002  |  Pubmed ID: 12205449

Transplanted Neural Stem Cells Survive, Differentiate, and Improve Neurological Motor Function After Experimental Traumatic Brain Injury

Neurosurgery. Oct, 2002  |  Pubmed ID: 12234415

Using the neural stem cell (NSC) clone C17.2, we evaluated the ability of transplanted murine NSCs to attenuate cognitive and neurological motor deficits after traumatic brain injury.

Neural Stem Cells Display an Inherent Mechanism for Rescuing Dysfunctional Neurons

Nature Biotechnology. Nov, 2002  |  Pubmed ID: 12379867

We investigated the hypothesis that neural stem cells (NSCs) possess an intrinsic capacity to "rescue" dysfunctional neurons in the brains of aged mice. The study focused on a neuronal cell type with stereotypical projections that is commonly compromised in the aged brain-the dopaminergic (DA) neuron. Unilateral implantation of murine NSCs into the midbrains of aged mice, in which the presence of stably impaired but nonapoptotic DA neurons was increased by treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), was associated with bilateral reconstitution of the mesostriatal system. Functional assays paralleled the spatiotemporal recovery of tyrosine hydroxylase (TH) and dopamine transporter (DAT) activity, which, in turn, mirrored the spatiotemporal distribution of donor-derived cells. Although spontaneous conversion of donor NSCs to TH(+) cells contributed to nigral reconstitution in DA-depleted areas, the majority of DA neurons in the mesostriatal system were "rescued" host cells. Undifferentiated donor progenitors spontaneously expressing neuroprotective substances provided a plausible molecular basis for this finding. These observations suggest that host structures may benefit not only from NSC-derived replacement of lost neurons but also from the "chaperone" effect of some NSC-derived progeny.

The Injured Brain Interacts Reciprocally with Neural Stem Cells Supported by Scaffolds to Reconstitute Lost Tissue

Nature Biotechnology. Nov, 2002  |  Pubmed ID: 12379868

Hypoxic-ischemic injury is a prototype for insults characterized by extensive tissue loss. Seeding neural stem cells (NSCs) onto a polymer scaffold that was subsequently implanted into the infarction cavities of mouse brains injured by hypoxia-ischemia allowed us to observe the multiple reciprocal interactions that spontaneously ensue between NSCs and the extensively damaged brain: parenchymal loss was dramatically reduced, an intricate meshwork of many highly arborized neurites of both host- and donor-derived neurons emerged, and some anatomical connections appeared to be reconstituted. The NSC-scaffold complex altered the trajectory and complexity of host cortical neurites. Reciprocally, donor-derived neurons were seemingly capable of directed, target-appropriate neurite outgrowth (extending axons to the opposite hemisphere) without specific external instruction, induction, or genetic manipulation of host brain or donor cells. These "biobridges" appeared to unveil or augment a constitutive reparative response by facilitating a series of reciprocal interactions between NSC and host, including promoting neuronal differentiation, enhancing the elaboration of neural processes, fostering the re-formation of cortical tissue, and promoting connectivity. Inflammation and scarring were also reduced, facilitating reconstitution.

Neural Stem Cells Spontaneously Express Dopaminergic Traits After Transplantation into the Intact or 6-hydroxydopamine-lesioned Rat

Experimental Neurology. Sep, 2002  |  Pubmed ID: 12429210

The ability to differentiate neural stem cells (NSCs) into dopamine neurons is fundamental to their role in cell replacement therapies for neurodegenerative disorders such as Parkinson's disease. We show here that when a clonal line (C17.2) of undifferentiated NSCs is transplanted into the intact or 6-hydroxydopamine-lesioned striatum, cells withdraw from the cell cycle (BrdU(-)), migrate extensively in the host striatum, and express markers associated with neuronal (beta-tubulin III(+), NSE(+), NeuN(+)) but not glial (GFAP(-), MBP(-), A2B5(-)) differentiation. Importantly, by 2-5 weeks postgrafting, in the majority of these transplants, nearly all engrafted cells express the dopamine-synthesizing enzymes tyrosine hydroxylase and aromatic L-amino decarboxylase, sometimes resulting in changes in motor behavior. In contrast, no NSCs stain for dopamine-beta-hydroxylase, choline acetyltransferase, glutamic acid decarboxylase, or serotonin. We conclude that, following transplantation into the intact or 6-hydroxydopamine-lesioned rat, the adult brain contains intrinsic cues sufficient to direct the specific expression of dopaminergic traits in immature multipotential neural stem cells.

Neural Stem Cells for CNS Repair: State of the Art and Future Directions

Advances in Experimental Medicine and Biology. 2002  |  Pubmed ID: 12614070

Genetic Programs and Responses of Neural Stem/progenitor Cells During Demyelination: Potential Insights into Repair Mechanisms in Multiple Sclerosis

Physiological Genomics. Aug, 2003  |  Pubmed ID: 12923300

In recent years, it has become evident that the adult mammalian CNS contains a population of neural stem cells (NSCs) described as immature, undifferentiated, multipotent cells, that may be called upon for repair in neurodegenerative and demyelinating diseases. NSCs may give rise to oligodendrocyte progenitor cells (OPCs) and other myelinating cells. This article reviews recent progress in elucidating the genetic programs and dynamics of NSC and OPC proliferation, differentiation, and apoptosis, including the response to demyelination. Emerging knowledge of the molecules that may be involved in such responses may help in the design of future stem cell-based treatment of demyelinating diseases such as multiple sclerosis.

Neural Stem Cell Biology May Be Well Suited for Improving Brain Tumor Therapies

Cancer Journal (Sudbury, Mass.). May-Jun, 2003  |  Pubmed ID: 12952304

Neural stem cells (NSCs) are capable of tremendous migratory potential to areas of pathology in the central nervous system. When implanted into a diseased or injured nervous system, NSCs can travel through great distances to and engraft within areas of discrete as well as diffuse abnormalities. Engraftment is often followed by integration into the local neural milieu, accompanied by stable gene expression from the NSCs. In addition, the pluripotency of NSCs endows them with the capability to replace diseased tissues in an appropriate manner. Recent evidence has also suggested that engrafted exogenous NSCs may have effects on the surrounding microenvironment, such as promoting protection and/or regeneration of host neural pathways. These characteristics of NSCs would seem to make them ideal agents for the treatment of various central nervous system pathologies, especially brain tumors. Brain tumors are generally difficult to treat because of the unique location of the lesions. In primary gliomas, the extensive infiltrative nature of the tumor cells presents a challenge for their effective and total eradication, hence the high rate of treatment failure and disease recurrence. In addition, normal brain structures are distorted and are often destroyed by the growing neoplasm. Even with effective therapy to surgically resect and destroy the neoplastic tissues, the brain is still injured, which often leaves the patient in a debilitated state. The unique ability of NSCs to "home in" on tumor cells followed by the delivery of a desired gene product makes the NSC a very promising agent in brain tumor therapy. Cytolytic viruses and genes coding for anti-tumor cytokines, pro-drug converting enzymes, and various neurotrophic factors have all been engineered into engraftable NSCs for delivery to tumors. When they are specially tagged, such injected NSCs can be visualized with the use of novel imaging techniques and tracked in vivo within living animals over real time. If the NSCs were also capable of participating in the subsequent repair and regeneration of the tumor-afflicted brain-at present a potential but as-yet-unproven aspect of this intervention-then its role in abetting anti-tumor therapy would be complete. It is important to emphasize, however, that the use of NSCs is adjunctive and is not a replacement for other therapies that should be used in parallel.

Realistic Prospects for Stem Cell Therapeutics

Hematology / the Education Program of the American Society of Hematology. American Society of Hematology. Education Program. 2003  |  Pubmed ID: 14633792

Studies of the regenerating hematopoietic system have led to the definition of many of the fundamental principles of stem cell biology. Therapies based on a range of tissue stem cells have been widely touted as a new treatment modality, presaging an emerging new specialty called regenerative medicine that promises to harness stem cells from embryonic and somatic sources to provide replacement cell therapies for genetic, malignant, and degenerative conditions. Insights borne from stem cell biology also portend development of protein and small molecule therapeutics that act on endogenous stem cells to promote repair and regeneration. Much of the newfound enthusiasm for regenerative medicine stems from the hope that advances in the laboratory will be followed soon thereafter by breakthrough treatments in the clinic. But how does one sort through the hype to judge the true promise? Are stem cell biologists and the media building expectations that cannot be met? Which diseases can be treated, and when can we expect success? In this review, we outline the realms of investigation that are capturing the most attention, and consider the current state of scientific understanding and controversy regarding the properties of embryonic and somatic (adult) stem cells. Our objective is to provide a framework for appreciating the promise while at the same time understanding the challenges behind translating fundamental stem cell biology into novel clinical therapies.

Constitutive EGFR Signaling Confers a Motile Phenotype to Neural Stem Cells

Molecular and Cellular Neurosciences. Dec, 2003  |  Pubmed ID: 14697673

The epidermal growth factor receptor (EGFR) has been shown to play an important role in brain development, including stem and precursor cell survival, proliferation, differentiation, and migration. To further examine the temporal and spatial requirements of erbB signals in uncommitted neural stem cells (NSCs), we expressed the ligand-independent EGF receptor, EGFRvIII, in C17.2 NSCs. These NSCs are known to migrate and to evince a tropic response to neurodegenerative environments in vivo but for which an underlying mechanism remains unclear. We show that enhanced erbB signaling via constitutive kinase activity of EGFRvIII in NSCs sustains an immature phenotype and enhances NSC migration.

Fate of Multipotent Neural Precursor Cells Transplanted into Mouse Retina Selectively Depleted of Retinal Ganglion Cells

Experimental Neurology. Mar, 2004  |  Pubmed ID: 14980806

In some parts of the CNS, depletion of a particular class of neuron might induce changes in the microenvironment that influence the differentiation of newly grafted neural precursor cells. This hypothesis was tested in the retina by inducing apoptotic retinal ganglion cell (RGC) death in neonatal and adult female mice and examining whether intravitreally grafted male neural precursor cells (C17.2), a neural stem cell (NSC)-like clonal line, become incorporated into these selectively depleted retinae. In neonates, rapid RGC death was induced by removal of the contralateral superior colliculus (SC), in adults, delayed RGC death was induced by unilateral optic nerve (ON) transection. Cells were injected intravitreally 6-48 h after SC ablation (neonates) or 0-7 days after ON injury (adults). Cells were also injected into non-RGC depleted neonatal and adult retinae. At 4 or 8 weeks, transplanted cells were identified using a Y-chromosome marker and in situ hybridisation or by their expression of the lacZ reporter gene product Escherichia coli beta-galactosidase (beta-gal). No C17.2 cells were identified in axotomised adult-injected eyes undergoing delayed RGC apoptosis (n = 16). Donor cells were however stably integrated within the retina in 29% (15/55) of mice that received C17.2 cell injections 24 h after neonatal SC ablation; 6-31% of surviving cells were found in the RGC layer (GCL). These NSC-like cells were also present in intact retinae, but on average, there were fewer cells in GCL. In SC-ablated mice, most grafted cells did not express retinal-specific markers, although occasional donor cells in the GCL were immunopositive for beta-III tubulin, a protein highly expressed by, but not specific to, developing RGCs. Targeted rapid RGC depletion thus increased cell incorporation into the GCL, but grafted C17.2 cells did not appear to differentiate into an RGC phenotype.

Minocycline Inhibits Contusion-triggered Mitochondrial Cytochrome C Release and Mitigates Functional Deficits After Spinal Cord Injury

Proceedings of the National Academy of Sciences of the United States of America. Mar, 2004  |  Pubmed ID: 14981254

We investigated whether permeability transition-mediated release of mitochondrial cytochrome c is a potential therapeutic target for treating acute spinal cord injury (SCI). Based on previous reports, minocycline, a second-generation tetracycline, exerts neuroprotection partially by inhibiting mitochondrial cytochrome c release and reactive microgliosis. We first evaluated cytochrome c release at the injury epicenter after a T10 contusive SCI in rats. Cytochrome c release peaked at approximately 4-8 h postinjury. A dose-response study generated a safe pharmacological regimen that enabled i.p. minocycline to significantly lower cytosolic cytochrome c at the epicenter 4 h after SCI. In the long-term study, i.p. minocycline (90 mg/kg administered 1 h after SCI followed by 45 mg/kg administered every 12 h for 5 days) markedly enhanced long-term hind limb locomotion relative to that of controls. Coordinated motor function and hind limb reflex recoveries also were improved significantly. Histopathology suggested that minocycline treatment alleviated later-phase tissue loss, with significant sparing of white matter and ventral horn motoneurons at levels adjacent to the epicenter. Furthermore, glial fibrillary acidic protein and 2',3' cyclic nucleotide 3' phosphodiesterase immunocytochemistry showed an evident reduction in astrogliosis and enhanced survival of oligodendrocytes. Therefore, release of mitochondrial cytochrome c is an important secondary injury mechanism in SCI. Drugs with multifaceted effects in antagonizing this process and microgliosis may protect a proportion of spinal cord tissue that is clinically significant for functional recovery. Minocycline, with its proven clinical safety, capability to cross the blood-brain barrier, and demonstrated efficacy during a clinically relevant therapeutic window, may become an effective therapy for acute SCI.

Reality and Immortality--neural Stem Cells for Therapies

Nature Biotechnology. Mar, 2004  |  Pubmed ID: 14990948

Taking Stock and Planning for the Next Decade: Realistic Prospects for Stem Cell Therapies for the Nervous System

Journal of Neuroscience Research. Apr, 2004  |  Pubmed ID: 15048913

In thinking about the practical application of stem cell biology to clinical situations--particularly for the central nervous system (CNS)-it is instructive to remember that the neural stem cell (NSC) field--as a prototype for somatic stem cells in general-emerged as the unanticipated byproduct of investigations by developmental neurobiologists into fundamental aspects of neural determination, commitment, and plasticity. Stem cell behavior is ultimately an expression of developmental principles, an alluring vestige from the more plastic and generative stages of organogenesis. In attempting to apply stem cell biology therapeutically, it is instructive always to bear in mind what role the stem cell plays in development and to what cues it was "designed" to respond in trying to understand the "logic" behind its behavior (both what investigators want to see and what investigators do not want to see). Furthermore, in transplantation paradigms, the interaction between engrafted NSCs and recipient host is a dynamic, complex, ongoing reciprocal interaction where both entities are constantly in flux. In this review, we propose a "roadmap" to the clinic, with a particular emphasis on flagging the "potholes" and "speed bumps" through which we must navigate. Despite the admonitions to be circumspect, we also suggest disease processes that may be within the grasp of proven stem cell properties and might be approachable in the relatively near future.

Neural Stem/progenitor Cells Express Costimulatory Molecules That Are Differentially Regulated by Inflammatory and Apoptotic Stimuli

The American Journal of Pathology. May, 2004  |  Pubmed ID: 15111308

Increased expression of the costimulatory molecule CD80 (B7-1) was noted in the subventricular zone of the brain during the course of experimental autoimmune encephalomyelitis (EAE). This area of the brain is a neural stem cell (NSC) niche in the adult. We show that isolated NSCs from adult brain express CD80 and CD86 (B7-2) and this expression is increased after exposure to IFN-gamma or TNF-alpha, the prototypical Th1 cytokines expressed during EAE. CD80 and CD86 expressed by NSCs are functional and can costimulate allogeneic cells in a mixed lymphocyte reaction. Furthermore, cross-linking of CD80 on the surface of NSCs results in apoptosis of NSCs. In vitro, we show that T cells can interact with NSCs and form conjugates with redistribution of CD3 on the surface of T cells to the area of contact. These data raise the possibility that during CNS inflammatory diseases such as EAE, NSCs may express immune molecules and interact with the inflammatory environment potentially resulting in injury to the NSCs, which may have implications for repair mechanisms in the central nervous system.

Stem Cells: Cross-talk and Developmental Programs

Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. May, 2004  |  Pubmed ID: 15293810

The thesis advanced in this essay is that stem cells-particularly those in the nervous system-are components in a series of inborn 'programs' that not only ensure normal development, but persist throughout life so as to maintain homeostasis in the face of perturbations-both small and great. These programs encode what has come to be called 'plasticity'. The stem cell is one of the repositories of this plasticity. This review examines the evidence that interaction between the neural stem cell (as a prototypical somatic stem cell) and the developing or injured brain is a dynamic, complex, ongoing reciprocal set of interactions where both entities are constantly in flux. We suggest that this interaction can be viewed almost from a 'systems biology' vantage point. We further advance the notion that clones of exogenous stem cells in transplantation paradigms may not only be viewed for their therapeutic potential, but also as biological tools for 'interrogating' the normal or abnormal central nervous system environment, indicating what salient cues (among the many present) are actually guiding the expression of these 'programs'; in other words, using the stem cell as a 'reporter cell'. Based on this type of analysis, we suggest some of the relevant molecular pathways responsible for this 'cross-talk' which, in turn, lead to proliferation, migration, cell genesis, trophic support, protection, guidance, detoxification, rescue, etc. This type of developmental insight, we propose, is required for the development of therapeutic strategies for neurodegenerative disease and other nervous system afflictions in humans. Understanding the relevant molecular pathways of stem cell repair phenotype should be a priority, in our view, for the entire stem cell field.

Differentiation and Tropic/trophic Effects of Exogenous Neural Precursors in the Adult Spinal Cord

The Journal of Comparative Neurology. Nov, 2004  |  Pubmed ID: 15514921

The fate of exogenous neural stem cells (NSCs) in the environment of the adult nervous system continues to be a matter of debate. In the present study, we report that cells of the murine NSC clone C17.2, when grafted into the lumbar segments of the spinal cord of adult rats, survive and undergo partial differentiation. C17.2 cells migrate avidly toward axonal tracts and nerve roots and differentiate into nonmyelinating ensheathing cells. Notably, C17.2 cells induce the de novo formation of host axon tracts aiming at graft innervation. Differentiation and inductive properties of C17.2 cells are independent of the presence of lesions in the spinal cord. The tropic/trophic interactions of C17.2 NSCs with host axons, the avid C17.2 cell-host axon contacts, and the ensheathing properties of these cells are related to their complex molecular profile, which includes the expression of trophic cytokines and neurotrophins such as glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor, glial growth factor receptors such as ErbB-2; and PASK, the mammalian homologue of the fray gene that is involved in axon ensheathment. These results show that NSCs might not only play a critical supportive role in repairing axonal injury in the adult spinal cord but also can be used as probes for exploring the molecular underpinnings of the regenerative potential of the mature nervous system after injury.

Neural Stem Cells Protect Against Glutamate-induced Excitotoxicity and Promote Survival of Injured Motor Neurons Through the Secretion of Neurotrophic Factors

Molecular and Cellular Neurosciences. Nov, 2004  |  Pubmed ID: 15519246

Besides their capacity to give rise to neurons and/or glia, neural stem cells (NSCs) appear to inherently secrete neurotrophic factors beneficial to injured neurons. To test this potential, we have implanted NSCs onto or adjacent to spinal cord cultures. When NSCs were placed adjacent to the spinal cord sections, motor neuron axons grew toward the NSCs. Furthermore, conditioned medium from NSCs cultures was also able to induce similar axonal outgrowth, suggesting that these NSCs secrete soluble factors that have tropic and/or trophic properties. ELISA revealed that the NSCs secrete glial cell-line-derived factor (GDNF) and nerve growth factor (NGF). Interestingly, preincubation of the conditioned medium with GDNF-blocking antibodies abolished axonal outgrowth. We also showed that NSCs can protect spinal cord cultures from experimentally induced excitotoxic damage. The neuroprotective potential of NSCs was further confirmed in vivo by their ability to protect against motor neuron cell death.

Directed Migration of Neural Stem Cells to Sites of CNS Injury by the Stromal Cell-derived Factor 1alpha/CXC Chemokine Receptor 4 Pathway

Proceedings of the National Academy of Sciences of the United States of America. Dec, 2004  |  Pubmed ID: 15608062

Migration toward pathology is the first critical step in stem cell engagement during regeneration. Neural stem cells (NSCs) migrate through the parenchyma along nonstereotypical routes in a precise directed manner across great distances to injury sites in the CNS, where they might engage niches harboring local transiently expressed reparative signals. The molecular mechanisms for NSC mobilization have not been identified. Because NSCs seem to home similarly to pathologic sites derived from disparate etiologies, we hypothesized that the inflammatory response itself, a characteristic common to all, guides the behavior of potentially reparative cells. As proof of concept, we show that human NSCs migrate in vivo (including from the contralateral hemisphere) toward an infarcted area (a representative CNS injury), where local astrocytes and endothelium up-regulate the inflammatory chemoattractant stromal cell-derived factor 1alpha (SDF-1alpha). NSCs express CXC chemokine receptor 4 (CXCR4), the cognate receptor for SDF-1alpha. Exposure of SDF-1alpha to quiescent NSCs enhances proliferation, promotes chain migration and transmigration, and activates intracellular molecular pathways mediating engagement. CXCR4 blockade abrogates their pathology-directed chain migration, a developmentally relevant mode of tangential migration that, if recapitulated, could explain homing along nonstereotypical paths. Our data implicate SDF-1alpha/CXCR4, representative of the inflammatory milieu characterizing many pathologies, as a pathway that activates NSC molecular programs during injury and suggest that inflammation may be viewed not simply as playing an adverse role but also as providing stimuli that recruit cells with a regenerative homeostasis-promoting capacity. CXCR4 expression within germinal zones suggests that NSC homing after injury and migration during development may invoke similar mechanisms.

A Review and Rationale for the Use of Cellular Transplantation As a Therapeutic Strategy for Traumatic Brain Injury

Journal of Neurotrauma. Nov, 2004  |  Pubmed ID: 15684646

Experimental research during the past decade has greatly increased our understanding of the pathophysiology of traumatic brain injury (TBI) and allowed us to develop neuroprotective pharmacological therapies. Encouraging results of experimental pharmacological interventions, however, have not been translated into successful clinical trials, to date. Traumatic brain injury is now believed to be a progressive degenerative disease characterized by cell loss. The limited capacity for self-repair of the brain suggests that functional recovery following TBI is likely to require cellular transplantation of exogenous cells to replace those lost to trauma. Recent advances in central nervous system transplantation techniques involve technical and experimental refinements and the analysis of the feasibility and efficacy of transplantation of a range of stem cells, progenitor cells and postmitotic cells. Cellular transplantation has begun to be evaluated in several models of experimental TBI, with promising results. The following is a compendium of these new and exciting studies, including a critical discussion of the rationale and caveats associated with cellular transplantation techniques in experimental TBI research. Further refinements in future research are likely to improve results from transplantation-based treatments for TBI.

Neural Stem Cells Implanted into MPTP-treated Monkeys Increase the Size of Endogenous Tyrosine Hydroxylase-positive Cells Found in the Striatum: a Return to Control Measures

Cell Transplantation. 2005  |  Pubmed ID: 15929553

Neural stem cells (NSC) have been shown to migrate towards damaged areas, produce trophic factors, and replace lost cells in ways that might be therapeutic for Parkinson's disease (PD). However, there is very little information on the effects of NSC on endogenous cell populations. In the current study, effects of implanted human NSC (hNSC) on endogenous tyrosine hydroxylase-positive cells (TH+ cells) after treatment with 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine (MPTP) were explored in nonhuman primates. After MPTP damage and in PD, the primate brain is characterized by decreased numbers of dopamine neurons in the substantia nigra (SN) and an increase in neurons expressing TH in the caudate nucleus. To determine how implanted NSC might affect these cell populations, 11 St. Kitts African green monkeys were treated with the selective dopaminergic neurotoxin, MPTP. Human NSC were implanted into the left and right caudate nucleus and the right SN of eight of the MPTP-treated monkeys. At either 4 or 7 months after NSC implants, the brains were removed and the size and number of TH+ cells in the target areas were assessed. The results were compared to data obtained from normal untreated control monkeys and to the three unimplanted MPTP-treated monkeys. The majority of hNSC were found bilaterally along the nigrostriatal pathway and in the substantia nigra, while relatively few were found in the caudate. In the presence of NSC, the number and size of caudate TH+ cells returned to non-MPTP-treated control levels. MPTP-induced and hNSC-induced changes in the putamen were less apparent. We conclude that after MPTP treatment in the primate, hNSC prevent the MPTP-induced upregulation of TH+ cells in the caudate and putamen, indicating that hNSC may be beneficial to maintaining a normal striatal environment.

Expression Profile of an Operationally-defined Neural Stem Cell Clone

Experimental Neurology. Aug, 2005  |  Pubmed ID: 15992799

Neural stem cells (NSCs) are the most primordial and least committed cells of the nervous system, the cells that exist before regional specification develops. Because immunocytochemically-detectable markers that are sufficiently specific and sensitive to define an NSC have not yet been fully defined, we have taken the strong view that, to be termed a "stem cell" in the nervous system--in contrast to a "progenitor" or "precursor" (whose lineage commitment is further restricted)--a single neuroectodermally-derived cell must fulfill an operational definition that is essentially similar to that used in hematopoiesis. In other words, it must possess the following functional properties: (1) "Multipotency", i.e., the ability to yield mature cells in all three fundamental neural lineages throughout the nervous system--neurons (of all subtypes), astrocytes (of all types), oligodendrocytes--in multiple regional and developmental contexts and in a region and developmental stage-appropriate manner. (2) The ability to populate a developing region and/or repopulate an ablated or degenerated region of the nervous system with appropriate cell types. (3) The ability to be serially transplanted. (4) "Self-renewal", i.e., the ability to produce daughter cells (including new NSCs) with identical properties and potential. Having identified a murine neural cell clone that fulfills this strict operational definition--in contrast to other studies that used less rigorous or non-operational criteria for defining an NSC (e.g., the "neurosphere" assay)--we then examined, by comparing gene expression profiles, the relationship such a cell might have to (a) a multipotent somatic stem cell from another organ system (the hematopoietic stem cell [HSC]); (b) a pluripotent stem cell derived from the inner cell mass and hence without organ assignment (an embryonic stem cell); (c) neural cells isolated and maintained primarily as neurospheres but without having been subjected to the above mentioned operational screen ("CNS-derived neurospheres"). ESCs, HSCs, and operationally-defined NSCs--all of which have been identified not only by markers but by functional assays in their respective systems and whose state of differentiation could be synchronized--shared a large number of genes. Although, as expected, the most stem-like genes were expressed by ESCs, NSCs and HSCs shared a number of genes. CNS-derived neurospheres, on the other hand, expressed fewer "stem-like" genes held in common by the other operationally-defined stem cell populations. Rather they displayed a profile more consistent with differentiated neural cells. (Genes of neural identity were shared with the NSC clone.) Interestingly, when the operationally-defined NSC clone was cultured as a neurosphere (rather than in monolayer), its expression pattern shifted from a "stem-like" pattern towards a more "differentiated" one, suggesting that the neurosphere, without functional validation, may be a poor model for predicting stem cell attributes because it consists of heterogeneous populations of cells, only a small proportion of which are truly "stem-like". Furthermore, when operational definitions are employed, a common set of stem-like genes does emerge across both embryonic and somatic stem cells of various organ systems, including the nervous system.

A Role for Stem Cell Biology in the Physiological and Pathological Aspects of Aging

Journal of the American Geriatrics Society. Sep, 2005  |  Pubmed ID: 16131354

The stem cell is an essential component of a developmental phenomenon-one of the key components of a program fundamental to organogenesis and the maintenance of homeostasis throughout life. The stem cell confers plasticity to the process. Because aging is part of the developmental arc, an understanding of the stem cell is essential to understanding aging and some of the degenerative conditions that accompany it. Because stem cells may be isolated and manipulated ex vivo and then reimplanted into organs, they may serve a variety of functions. They have the capability of migrating long distances and targeting pathological conditions, of expressing therapeutic genes and responding to cues that shift their differentiation toward deficient lineages. Therefore, stem cells may be used for cell replacement, for therapeutic interventions, and potentially to modify aging.

Caspase-mediated Cell Death Predominates Following Engraftment of Neural Progenitor Cells into Traumatically Injured Rat Brain

Brain Research. Dec, 2005  |  Pubmed ID: 16309635

Neural progenitor cells (NPCs) have been shown to be a promising therapy for cell replacement and gene transfer in neurological diseases including traumatic brain injury (TBI). However, NPCs often survive poorly after transplantation despite immunosuppression, and the mechanisms of graft cell death are unknown. In this study, we evaluated caspase- and calpain-mediated mechanisms of cell death of neonatal mouse C17.2 progenitor cells, transplanted at 24 h following lateral fluid percussion brain injury (FP) in rats. Adult Male Sprague-Dawley rats (n = 30) were subjected to lateral FP injury (n = 18) or sham surgery (n = 12). C17.2 cells labeled with green fluorescent dye (CMFDA) were engrafted in the perilesional deep cortex, and animals were sacrificed at 24 h, 72 h and 1 week post-transplantation. Pro-apoptotic caspase-mediated cleavage products (Ab246) and calpain-mediated cleavage products (Ab38) were detected in the engrafted cells using immunohistochemistry. Only 2 to 4.5% of grafted NPCs were found to survive at 24 h post-transplantation, regardless of injury status of the host brain, although brain-injured animals had significantly fewer graft cells than sham-injured animals. Limited caspase and calpain-mediated graft cell death was observed in both sham- and brain-injured animals, and caspase-mediated graft cell death was significantly greater than calpain-mediated graft cell death in all animals. Brain-injured animals had significantly increased caspase-mediated graft cell death compared to sham-injured animals. These results suggest that both the caspase and calpain family of proteases are involved in graft cell death, and that caspase-mediated apoptotic graft cell death predominates in the acute post-traumatic period following TBI.

Exploring Common Ground in the Stem Cell Ethical Debate--a Perspective for Scientists: Introduction to This Special Issue of Stem Cell Reviews

Stem Cell Reviews. Dec, 2005  |  Pubmed ID: 17142867

Immunomodulatory Neural Stem Cells for Brain Tumour Therapy

Expert Opinion on Biological Therapy. Dec, 2006  |  Pubmed ID: 17223735

Advances in the understanding of stem cells have enabled the development of novel therapies for brain tumours. Neural stem cells (NSCs) possess the ability to migrate throughout the CNS. By exploiting the tropism of NSCs to various neural pathologies (e.g., glioma, degeneration, stroke and so on) and the delivery of various immunomodulatory cytokines, new treatments for brain tumours have been investigated. These new strategies offer significantly more specificity than existing treatment regimens, such as surgery, radiation and chemotherapy. As methods in isolating and culturing NSCs are better understood, clinical applications of this therapeutic strategy may inevitably emerge. Here, the preclinical advances and the results supporting the effectiveness of stem cell therapies are reviewed. In addition, the obstacles to clinical development and methods to circumvent these caveats are discussed.

Neural Stem Cell Transplant Survival in Brains of Mice: Assessing the Effect of Immunity and Ischemia by Using Real-time Bioluminescent Imaging

Radiology. Dec, 2006  |  Pubmed ID: 17114629

To use bioluminescent imaging in a murine transplant model to monitor the in vivo responses of transplanted luciferase-gene-positive neural progenitor cells (NPCs) to host immunity and ischemia.

Physical Activity-mediated Functional Recovery After Spinal Cord Injury: Potential Roles of Neural Stem Cells

Regenerative Medicine. Nov, 2006  |  Pubmed ID: 17465758

As data elucidating the complexity of spinal cord injury pathophysiology emerge, it is increasingly being recognized that successful repair will probably require a multifaceted approach that combines tactics from various biomedical disciplines, including pharmacology, cell transplantation, gene therapy and material sciences. Recently, new evidence highlighting the benefit of physical activity and rehabilitation interventions during the post-injury phase has provided novel possibilities in realizing effective repair after spinal cord injury. However, before a comprehensive therapeutic strategy that optimally utilizes the benefits of each of these disciplines can be designed, the basic mechanisms by which these various interventions act must be thoroughly explored and important synergistic and antagonistic interactions identified. In examining the mechanisms by which physical activity-based functional recovery after spinal cord injury is effected, endogenous neural stem cells, in our opinion, engender a potentially key role. Multipotent neural stem cells possess many faculties that abet recovery, including the ability to assess the local microenvironment and deliver biofactors that promote neuroplasticity and regeneration, as well as the potential to replenish damaged or eradicated cellular elements. Encouragingly, the functional recovery owing to physical activity-based therapies appears relatively robust, even when therapy is initiated in the chronic stage of spinal cord injury. In this article, we review experimental outcomes related to our hypothesis that endogenous neural stem cells mediate the functional recovery noted in spinal cord injury following physical activity-based treatments. Overall, the data advocates the incorporation of increased physical activity as a component of the multidimensional treatment of spinal cord injury and underscores the critical need to employ research-based mechanistic approaches for developing future advances in the rehabilitation of neurological injury and disorders.

Large Animal Models Are Critical for Rationally Advancing Regenerative Therapies

Regenerative Medicine. Jul, 2006  |  Pubmed ID: 17465832

Gene Therapy: Can Neural Stem Cells Deliver?

Nature Reviews. Neuroscience. Jan, 2006  |  Pubmed ID: 16371952

Neural stem cells are a self-renewing population that generates the neurons and glia of the developing brain. They can be isolated, proliferated, genetically manipulated and differentiated in vitro and reintroduced into a developing, adult or pathologically altered CNS. Neural stem cells have been considered for use in cell replacement therapies in various neurodegenerative diseases, and an unexpected and potentially valuable characteristic of these cells has recently been revealed--they are highly migratory and seem to be attracted to areas of brain pathology such as ischaemic and neoplastic lesions. Here, we speculate on the ways in which neural stem cells might be exploited as delivery vehicles for gene therapy in the CNS.

Therapeutic Effects of Clenbuterol in a Murine Model of Amyotrophic Lateral Sclerosis

Neuroscience Letters. Apr 10-17, 2006  |  Pubmed ID: 16388902

We investigated the effects of clenbuterol, a beta2-adrenoceptor agonist with known anabolic and neuroprotective properties, on G93A-SOD1 mice, a transgenic murine model of familial amyotrophic lateral sclerosis (ALS). Relative to saline-treated vehicle controls (0.2 ml/kg/day; i.p.), early pathologic G93A-SOD1 mice treated with clenbuterol (1.5 mg/kg/day; i.p.) demonstrated a delayed onset of hindlimb signs as measured by rotarod performance, slowed disease progression, as well as trends toward mitigated losses of lumbar motoneurons and body weight. Responses in female G93A-SOD1 mice were favorable to those of males, suggesting synergistic effects between clenbuterol and sex-specific factors. Overall, our data suggest that clenbuterol offers therapeutic effects on ALS-related neuromuscular degeneration.

Networks of Gold Nanoparticles and Bacteriophage As Biological Sensors and Cell-targeting Agents

Proceedings of the National Academy of Sciences of the United States of America. Jan, 2006  |  Pubmed ID: 16434473

Biological molecular assemblies are excellent models for the development of nanoengineered systems with desirable biomedical properties. Here we report an approach for fabrication of spontaneous, biologically active molecular networks consisting of bacteriophage (phage) directly assembled with gold (Au) nanoparticles (termed Au-phage). We show that when the phage are engineered so that each phage particle displays a peptide, such networks preserve the cell surface receptor binding and internalization attributes of the displayed peptide. The spontaneous organization of these targeted networks can be manipulated further by incorporation of imidazole (Au-phage-imid), which induces changes in fractal structure and near-infrared optical properties. The networks can be used as labels for enhanced fluorescence and dark-field microscopy, surface-enhanced Raman scattering detection, and near-infrared photon-to-heat conversion. Together, the physical and biological features within these targeted networks offer convenient multifunctional integration within a single entity with potential for nanotechnology-based biomedical applications.

Beyond Fraud--stem-cell Research Continues

The New England Journal of Medicine. Jan, 2006  |  Pubmed ID: 16436761

Can Science Resolve the Ethical Impasse in Stem Cell Research?

Nature Biotechnology. Apr, 2006  |  Pubmed ID: 16601717

Advances in our understanding of the control of early human embryonic development could offer solutions to the moral dilemmas associated with human embryonic stem cell research.

Neural Progenitor Cells Engineered to Secrete GDNF Show Enhanced Survival, Neuronal Differentiation and Improve Cognitive Function Following Traumatic Brain Injury

The European Journal of Neuroscience. Apr, 2006  |  Pubmed ID: 16630059

We sought to evaluate the potential of C17.2 neural progenitor cells (NPCs) engineered to secrete glial cell line-derived neurotrophic factor (GDNF) to survive, differentiate and promote functional recovery following engraftment into the brains of adult male Sprague-Dawley rats subjected to lateral fluid percussion brain injury. First, we demonstrated continued cortical expression of GDNF receptor components (GFRalpha-1, c-Ret), suggesting that GDNF could have a physiological effect in the immediate post-traumatic period. Second, we demonstrated that GDNF over-expression reduced apoptotic NPC death in vitro. Finally, we demonstrated that GDNF over-expression improved survival, promoted neuronal differentiation of GDNF-NPCs at 6 weeks, as compared with untransduced (MT) C17.2 cells, following transplantation into the perilesional cortex of rats at 24 h post-injury, and that brain-injured animals receiving GDNF-C17.2 transplants showed improved learning compared with those receiving vehicle or MT-C17.2 cells. Our results suggest that transplantation of GDNF-expressing NPCs in the acute post-traumatic period promotes graft survival, migration, neuronal differentiation and improves cognitive outcome following traumatic brain injury.

Purkinje Neuron Degeneration in Nervous (nr) Mutant Mice is Mediated by a Metabolic Pathway Involving Excess Tissue Plasminogen Activator

Proceedings of the National Academy of Sciences of the United States of America. May, 2006  |  Pubmed ID: 16682647

Purkinje neurons (PNs), the central cells in cerebellar circuitry and function, constitute a vulnerable population in many human genetic, malignant, hypoxic, and toxic diseases. In the nervous (nr) mutant mouse, the majority of PNs die in the fourth to fifth postnatal weeks, but the responsible molecules are unknown. We first disclose a remarkable increase in mRNA expression and protein concentration in the nr cerebellum of tissue plasminogen activator (tPA), a gene closely linked to the mapped but as-yet-uncloned nr locus. Evidence that excessive tPA triggers nr PN death was obtained with organotypic slice cultures expressing the nr PN phenotype, in which an inhibitor of tPA led to increased nr PN survival. An antagonist of protein kinase C, a downstream component in the tPA pathway, also increased nr PN survival. Additional downstream targets in the tPA pathway (the mitochondrial voltage-dependent anion channel, brain-derived neurotrophic factor, and neurotrophin 3) were also abnormal, in parallel with the alterations in PN mitochondrial morphology, dendritic growth, and synaptogenesis that culminate in nr PN death and motor incoordination. We thus propose a molecular pathway by which the excessive tPA in nr cerebellum mediates PN degeneration.

Neural Stem Cells As Novel Cancer Therapeutic Vehicles

European Journal of Cancer (Oxford, England : 1990). Jun, 2006  |  Pubmed ID: 16697638

The startling resemblance of many of the behaviours of brain tumours to the intrinsic properties of the neural stem/progenitor cell has triggered a recent dual interest in arming stem cells to track and help eradicate tumours and in viewing stem cell biology as somehow integral to the emergence and/or propagation of the neoplasm itself. These aspects are reviewed and discussed here.

Neural Stem Cells May Be Uniquely Suited for Combined Gene Therapy and Cell Replacement: Evidence from Engraftment of Neurotrophin-3-expressing Stem Cells in Hypoxic-ischemic Brain Injury

Experimental Neurology. May, 2006  |  Pubmed ID: 16714016

Previously, we reported that, when clonal neural stem cells (NSCs) were transplanted into brains of postnatal mice subjected to unilateral hypoxic-ischemic (HI) injury (optimally 3-7 days following infarction), donor-derived cells homed preferentially (from even distant locations) to and integrated extensively within the large ischemic areas that spanned the hemisphere. A subpopulation of NSCs and host cells, particularly in the penumbra, "shifted" their differentiation towards neurons and oligodendrocytes, the cell types typically damaged following asphyxia and least likely to regenerate spontaneously and in sufficient quantity in the "post-developmental" CNS. That no neurons and few oligodendrocytes were generated from the NSCs in intact postnatal cortex suggested that novel signals are transiently elaborated following HI to which NSCs might respond. The proportion of "replacement" neurons was approximately 5%. Neurotrophin-3 (NT-3) is known to play a role in inducing neuronal differentiation during development and perhaps following injury. We demonstrated that NSCs express functional TrkC receptors. Furthermore, the donor cells continued to express a foreign reporter transgene robustly within the damaged brain. Therefore, it appeared feasible that neuronal differentiation of exogenous NSCs (as well as endogenous progenitors) might be enhanced if donor NSCs were engineered prior to transplantation to (over)express a bioactive gene such as NT-3. A subclone of NSCs transduced with a retrovirus encoding NT-3 (yielding >90% neurons in vitro) was implanted into unilaterally asphyxiated postnatal day 7 mouse brain (emulating one of the common causes of cerebral palsy). The subclone expressed NT-3 efficiently in vivo. The proportion of NSC-derived neurons increased to approximately 20% in the infarction cavity and >80% in the penumbra. The neurons variously differentiated further into cholinergic, GABAergic, or glutamatergic subtypes, appropriate to the cortex. Donor-derived glia were rare, and astroglial scarring was blunted. NT-3 likely functioned not only on donor cells in an autocrine/paracrine fashion but also on host cells to enhance neuronal differentiation of both. Taken together, these observations suggest (1) the feasibility of taking a fundamental biological response to injury and augmenting it for repair purposes and (2) the potential use of migratory NSCs in some degenerative conditions for simultaneous combined gene therapy and cell replacement during the same procedure in the same recipient using the same cell (a unique property of cells with stem-like attributes).

Human Neurospheres Derived from the Fetal Central Nervous System Are Regionally and Temporally Specified but Are Not Committed

Experimental Neurology. May, 2006  |  Pubmed ID: 16714017

Proliferating single cells were isolated from various CNS regions (telencephalon, diencephalon, midbrain, cerebellum, pons and medulla, and spinal cord) of human fetal cadavers at 13 weeks of gestation and grown as neurospheres in long-term cultures. We investigated whether neural stem cells (NSCs) or progenitors within spheres have specific regional or temporal characteristics with regard to growth, differentiation, and region-specific gene expression, and whether these molecular specifications are reversible. Regardless of regional origin, all of the neurospheres were found to contain cells of different subtypes, which suggests that multipotent NSCs, progenitors or radial glial cells co-exist with restricted neuronal or glial progenitors within the neurospheres. Neurospheres from the forebrain grew faster and gave rise to significantly more neurons than did those from either the midbrain or hindbrain, and regional differences in neuronal differentiation appeared to be sustained during long-term passage of neurospheres in culture. There was also a trend towards a reduction in neuronal emergence from the respective neurospheres over time in culture, although the percentages of neurons generated from cerebellum-derived neurospheres increased dramatically. These results suggest that differences in neuronal differentiation for the various neurospheres are spatially and temporally determined. In addition, the properties of glial fibrillary acidic protein (GFAP)-, glutamate-, and gamma-aminobutyric acid (GABA)-expressing cells derived from neurospheres of the respective CNS regions appear to be regionally and temporally different. Isolated human neurospheres from different CNS compartments expressed distinctive molecular markers of regional identity and maintained these patterns of region-specific gene expression during long-term passage in vitro. To determine the potential of human neurospheres for regional fate plasticity, single spheres from the respective regions were co-cultured with embryonic day 16.5 (E16.5 d) mouse brain slices. Specific cues from the developing mouse brain tissues induced the human neurospheres to express different marker genes of regional identity and to suppress the expression of their original marker genes. Thus, even the early regional identities of human neurospheres may not be irreversible and may be altered by local inductive cues. These findings have important implications for understanding the characteristics of growth, differentiation, and molecular specification of human neurospheres derived from the developing CNS, as well as the therapeutic potential for neural repair.

Acute Injury Directs the Migration, Proliferation, and Differentiation of Solid Organ Stem Cells: Evidence from the Effect of Hypoxia-ischemia in the CNS on Clonal "reporter" Neural Stem Cells

Experimental Neurology. May, 2006  |  Pubmed ID: 16737696

Clonal neural cells with stem-like features integrate appropriately into the developing and degenerating central and peripheral nervous system throughout the neuraxis. In response to hypoxic-ischemic (HI) injury, previously engrafted, integrated, and quiescent clonal neural stem cells (NSCs) transiently re-enter the cell cycle, migrate preferentially to the site of ischemia, and differentiate into neurons and oligodendrocytes, the neural cell types typically lost following HI brain injury. They also replenish the supply of immature uncommitted resident stem/progenitor cells. Although they yield astrocytes, scarring is inhibited. These responses appear to occur most robustly within a 3-7 day "window" following HI during which signals are elaborated that upregulate genetic programs within the NSC that mediate proliferation, migration, survival, and differentiation, most of which appear to be terminated once the "window closes" and the chronic phase ensues, sending the NSCs into a quiescent state. These insights derived from using the stem cell in a novel role--as a "reporter" cell--to both track and probe the activity of endogenous stem cells as well as to "interrogate" and "report" the genes differentially induced by the acutely vs. chronically injured milieu. NSCs may be capable of the replacement of cells, genes, and non-diffusible factors in both a widespread or more circumscribed manner (depending on the therapeutic demands of the clinical situation). They may be uniquely responsive to some types of neurodegenerative conditions. We submit that these various capabilities are simply the normal expression of the basic homeostasis-preserving biologic properties and attributes of a stem cell which, if used rationally and in concert with this biology, may be exploited for therapeutic ends.

Special Issue: the Intersection of Stem/progenitor Cell Biology and Hypoxic-ischemic Cerebral Injury/stroke

Experimental Neurology. May, 2006  |  Pubmed ID: 16798271

Intrinsic Resistance of Neural Stem Cells to Toxic Metabolites May Make Them Well Suited for Cell Non-autonomous Disorders: Evidence from a Mouse Model of Krabbe Leukodystrophy

Journal of Neurochemistry. Jun, 2006  |  Pubmed ID: 16805770

While transplanted neural stem cells (NSCs) have been shown to hold promise for cell replacement in models of a number of neurological disorders, these examples have typically been under conditions where the host cells become dysfunctional due to a cell autonomous etiology, i.e. a 'sick' cell within a relatively supportive environment. It has long been held that cell replacement in a toxic milieu would not likely be possible; donor cells would succumb in much the same way as endogenous cells had. Many metabolic diseases are characterized by this situation, suggesting that they would be poor targets for cell replacement therapies. On the other hand, models of such diseases could prove ideal for testing the capacity for cell replacement under such challenging conditions. In the twitcher (twi ) mouse -- as in patients with Krabbe or globoid cell leukodystrophy (GLD), for which it serves as an authentic model -- loss of galactocerebrosidase (GalC) activity results in the accumulation of psychosine, a toxic glycolipid. Twi mice, like children with GLD, exhibit inexorable neurological deterioration presumably as a result of dysfunctional and ultimately degenerated oligodendrocytes with loss of myelin. It is believed that GLD pathophysiology is related to a psychosine-filled environment that kills not only host oligodendrocytes but theoretically any new cells placed into that milieu. Through the implantation of NSCs into the brains of both neonatal and juvenile/young adult twi mice, we have determined that widespread oligodendrocyte replacement and remyelination is feasible. NSCs appear to be intrinsically resistant to psychosine -- more so in their undifferentiated state than when directed ex vivo to become oligodendrocytes. This resistance can be enhanced by engineering the NSCs to over-express GalC. Some twi mice grafted with such engineered NSCs had thicker white tracts and lived 2-3 times longer than expected. While their brains had detectable levels of GalC, it was probably more significant that their psychosine levels were lower than in twi mice that died at a younger age. This concept of resistance based on differentiation state extended to human NSCs which could similarly survive within the twi brain. Taken together, these results suggest a number of points regarding cellular therapies against degenerative diseases with a prominent cell non-autonomous component: Cell replacement is possible if cells resistant to the toxic environment are employed. Furthermore, an important aspect of successful treatment will likely be not only cell replacement but also cross-correction of host cells to provide them with enzyme activity and hence resistance. While oligodendrocyte replacement alone was not a sufficient treatment for GLD (even when extensive), the replacement of both cells and molecules -- e.g. with NSCs that could both become oligodendrocytes and 'pumps' for GalC -- emerges as a promising basis for a multidisciplinary strategy. Most neurological disease are complex in this way and will likely require multifaceted approaches, perhaps with NSCs serving as the 'glue'.

Cryopreservation by Slow Cooling with DMSO Diminished Production of Oct-4 Pluripotency Marker in Human Embryonic Stem Cells

Cryobiology. Oct, 2006  |  Pubmed ID: 16839540

We tested a "standard" cryopreservation protocol (slow cooling with 10% DMSO) on the human embryonic stem cell (hESC) line H9 containing an Oct-4 (POU5F1) promoter-driven, enhanced green fluorescent protein (EGFP) reporter to monitor maintenance of pluripotency. Cells were cooled to -80 degrees C in cryovials and then transferred to a -80 degrees C freezer. Cells were held at -80 degrees C for 3 days ("short-term storage") or 3 months ("long-term storage"). Vials were thawed in a +36 degrees C water bath and cells were cultured for 3, 7, or 14 days. Propidium iodide (PI) was used to assess cell viability by flow cytometry. Control cells were passaged on the same day that the frozen cells were thawed. The majority of cells in control hESC cultures were Oct-4 positive and almost 99% of EGFP+ cells were alive as determined by exclusion of PI. In contrast, the frozen cells, even after 3 days of culture, contained only 50% live cells, and only 10% were EGFP-positive. After 7 days in culture, the proportion of dead cells decreased and there was an increase in the Oct-4-positive population but microscopic examination revealed large patches of EGFP-negative cells within clusters of colonies even after 14 days of culturing. After 3 months of storage at -80 degrees C the deleterious effect of freezing was even more pronounced: the samples regained a quantifiable number of EGFP-positive cells only after 7 days of culturing following thawing. It is concluded that new protocols and media are required for freezing hESC and safe storage at -80 degrees C as well as studies of the mechanisms of stress-related events associated with cell cryopreservation.

Neural Stem Cells Rescue Nervous Purkinje Neurons by Restoring Molecular Homeostasis of Tissue Plasminogen Activator and Downstream Targets

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Jul, 2006  |  Pubmed ID: 16870729

Neural stem cells (NSCs) offer special therapeutic prospects because they can be isolated from the CNS, expanded ex vivo, and re-implanted into diseased CNS where they not only migrate and differentiate according to cues from host tissue but also appear to be capable of affecting host cells. In nervous (nr) mutant mice Purkinje neuron (PN) mitochondria become abnormal by the second postnatal week, and a majority of PNs die in the fourth to fifth weeks. We previously identified in nr cerebellum a 10-fold increase in tissue plasminogen activator (tPA) as a key component of the mechanism causing nr PN death. Here we report that undifferentiated wild-type murine NSCs, when transplanted into the newborn nr cerebellar cortex, do not replace host PNs but contact imperiled PNs and support their mitochondrial function, dendritic growth, and synaptogenesis, subsequently leading to the rescue of host PNs and restoration of motor coordination. This protection of nr PNs also is verified by an in vitro organotypic slice model in which nr cerebellar slices are cocultured with NSCs. Most importantly, the integrated NSCs in young nr cerebellum rectify excessive tPA mRNA and protein to close to normal levels and protect the mitochondrial voltage-dependent anion channel and neurotrophins, downstream targets of the tPA/plasmin proteolytic system. This report demonstrates for the first time that NSCs can rescue imperiled host neurons by rectifying their gene expression, elevating somatic stem cell therapeutic potential beyond solely cell replacement strategy.

Defining the Actual Sensitivity and Specificity of the Neurosphere Assay in Stem Cell Biology

Nature Methods. Oct, 2006  |  Pubmed ID: 16990812

For more than a decade the 'neurosphere assay' has been used to define and measure neural stem cell (NSC) behavior, with similar assays now used in other organ systems and in cancer. We asked whether neurospheres are clonal structures whose diameter, number and composition accurately reflect the proliferation, self-renewal and multipotency of a single founding NSC. Using time-lapse video microscopy, coculture experiments with genetically labeled cells, and analysis of the volume of spheres, we observed that neurospheres are highly motile structures prone to fuse even under ostensibly 'clonal' culture conditions. Chimeric neurospheres were prevalent independent of ages, species and neural structures. Thus, the intrinsic dynamic of neurospheres, as conventionally assayed, introduces confounders. More accurate conditions (for example, plating a single cell per miniwell) will be crucial for assessing clonality, number and fate of stem cells. These cautions probably have implications for the use of 'cytospheres' as an assay in other organ systems and with other cell types, both normal and neoplastic.

Adhesive Interactions Between Human Neural Stem Cells and Inflamed Human Vascular Endothelium Are Mediated by Integrins

Stem Cells (Dayton, Ohio). Nov, 2006  |  Pubmed ID: 17071855

Understanding the mechanisms by which stem cells home precisely to regions of injury or degeneration is of importance to both basic and applied regenerative medicine. Optimizing regenerative processes may depend on identifying the range of molecules that subserve stem cell trafficking. The "rolling" of extravasating cells on endothelium under conditions of physiological flow is the first essential step in the homing cascade and determines cell adhesion and transmigration. Using a laminar flow chamber to simulate physiological shear stress, we explored an aspect of this process by using human neural stem cells (hNSCs). We observed that the interactions between hNSCs and tumor necrosis factor-alpha (TNF-alpha)-stimulated human endothelium (simulating an inflamed milieu) are mediated by a subclass of integrins--alpha2, alpha6, and beta1, but not alpha4, alphav, or the chemokine-mediated pathway CXCR4-stromal cell-derived factor-1alpha--suggesting not only that the mechanisms mediating hNSC homing via the vasculature differ from the mechanisms mediating homing through parenchyma, but also that each step invokes a distinct pathway mediating a specialized function in the hNSC homing cascade. (TNF-alpha stimulation also upregulates vascular cell adhesion molecule-1 expression on the hNSCs themselves and increases NSC-endothelial interactions.) The selective use of integrin subgroups to mediate homing of cells of neuroectodermal origin may also be used to ensure that cells within the systemic circulation are delivered to the pathological region of a given organ to the exclusion of other, perhaps undesired, organs.

The Leading Edge of Stem Cell Therapeutics

Annual Review of Medicine. 2007  |  Pubmed ID: 17100553

Stem cells, by virtue of their defining property of self-renewal, represent an unlimited source of potentially functional human cells for basic research and regenerative medicine. Having validated the feasibility of cell-based therapeutic strategies over the past decade, mostly through the use of rodent cells, the stem cell field has now embarked upon a detailed characterization of human cells. Recent progress has included improved cell culture conditions, long-term propagation, directed differentiation, and transplantation of both human embryonic and somatic stem cells. Continued progress in understanding basic human stem cell biology, combined with a better handle on the fundamental pathophysiology of human diseases one wishes to target (including the use of human stem cells in primate and other large animal models of human disease), should help to move this technology closer to clinical application.

Quo Vadis Brain Repair? A Long Axonal Journey in the Adult CNS

Cell Stem Cell. Oct, 2007  |  Pubmed ID: 18371370

Recently in Nature Neuroscience, Gaillard et al. (2007) study axonal projections from embryonic cortical explants grafted into acutely damaged adult motor cortex. After attempting to rule out fusion of donor tissue with pre-existing host circuitry, the authors report robust long-distance donor axonal projections and synaptic integration into target regions appropriate for the motor system.

A Novel, Immortal, and Multipotent Human Neural Stem Cell Line Generating Functional Neurons and Oligodendrocytes

Stem Cells (Dayton, Ohio). Sep, 2007  |  Pubmed ID: 17556596

The discovery and study of neural stem cells have revolutionized our understanding of the neurogenetic process, and their inherent ability to adopt expansive growth behavior in vitro is of paramount importance for the development of novel therapeutics based on neural cell replacement. Recent advances in high-throughput assays for drug development and gene discovery dictate the need for rapid, reproducible, long-term expansion of human neural stem cells (hNSCs). In this view, the complement of wild-type cell lines currently available is insufficient. Here we report the establishment of a stable human neural stem cell line (immortalized human NSCs [IhNSCs]) by v-myc-mediated immortalization of previously derived wild-type hNSCs. These cells demonstrate three- to fourfold faster proliferation than wild-type cells in response to growth factors but retain rather similar properties, including multipotentiality. By molecular biology, biochemistry, immunocytochemistry, fluorescence microscopy, and electrophysiology, we show that upon growth factor removal, IhNSCs completely downregulate v-myc expression, cease proliferation, and differentiate terminally into three major neural lineages: astrocytes, oligodendrocytes, and neurons. The latter are functional, mature cells displaying clear-cut morphological and physiological features of terminally differentiated neurons, encompassing mostly the GABAergic, glutamatergic, and cholinergic phenotypes. Finally, IhNSCs produce bona fide oligodendrocytes in fractions up to 20% of total cell number. This is in contrast to the negligible propensity of hNSCs to generate oligodendroglia reported so far. Thus, we describe an immortalized hNSC line endowed with the properties of normal hNSCs and suitable for developing the novel, reliable assays and reproducible high-throughput gene and drug screening that are essential in both diagnostics and cell therapy studies.

Behavioral Improvement in a Primate Parkinson's Model is Associated with Multiple Homeostatic Effects of Human Neural Stem Cells

Proceedings of the National Academy of Sciences of the United States of America. Jul, 2007  |  Pubmed ID: 17586681

Stem cells have been widely assumed to be capable of replacing lost or damaged cells in a number of diseases, including Parkinson's disease (PD), in which neurons of the substantia nigra (SN) die and fail to provide the neurotransmitter, dopamine (DA), to the striatum. We report that undifferentiated human neural stem cells (hNSCs) implanted into 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated Parkinsonian primates survived, migrated, and had a functional impact as assessed quantitatively by behavioral improvement in this DA-deficit model, in which Parkinsonian signs directly correlate to reduced DA levels. A small number of hNSC progeny differentiated into tyrosine hydroxylase (TH) and/or dopamine transporter (DAT) immunopositive cells, suggesting that the microenvironment within and around the lesioned adult host SN still permits development of a DA phenotype by responsive progenitor cells. A much larger number of hNSC-derived cells that did not express neuronal or DA markers was found arrayed along the persisting nigrostriatal path, juxtaposed with host cells. These hNSCs, which express DA-protective factors, were therefore well positioned to influence host TH+ cells and mediate other homeostatic adjustments, as reflected in a return to baseline endogenous neuronal number-to-size ratios, preservation of extant host nigrostriatal circuitry, and a normalizing effect on alpha-synuclein aggregation. We propose that multiple modes of reciprocal interaction between exogenous hNSCs and the pathological host milieu underlie the functional improvement observed in this model of PD.

Neural Stem Cells Injected into the Sound-damaged Cochlea Migrate Throughout the Cochlea and Express Markers of Hair Cells, Supporting Cells, and Spiral Ganglion Cells

Hearing Research. Oct, 2007  |  Pubmed ID: 17659854

Most cases of hearing loss are caused by the death or dysfunction of one of the many cochlear cell types. We examined whether cells from a neural stem cell line could replace cochlear cell types lost after exposure to intense noise. For this purpose, we transplanted a clonal stem cell line into the scala tympani of sound damaged mice and guinea pigs. Utilizing morphological, protein expression and genetic criteria, stem cells were found with characteristics of both neural tissues (satellite, spiral ganglion, and Schwann cells) and cells of the organ of Corti (hair cells, supporting cells). Additionally, noise-exposed, stem cell-injected animals exhibited a small but significant increase in the number of satellite cells and Type I spiral ganglion neurons compared to non-injected noise-exposed animals. These results indicate that cells of this neural stem cell line migrate from the scala tympani to Rosenthal's canal and the organ of Corti. Moreover, they suggest that cells of this neural stem cell line may derive some information needed from the microenvironment of the cochlea to differentiate into replacement cells in the cochlea.

Neural Stem Cell Implantation Extends Life in Niemann-Pick C1 Mice

Journal of Applied Genetics. 2007  |  Pubmed ID: 17666780

In order to evaluate the phenotypic effects of implanted neural stem cells (NSCs) in the mouse model of Niemann-Pick C (NPC) disease, we injected a well-characterized clone of murine NSCs into the cerebella of neonatal Npc1(-/-) and control mice. The implanted cells survived and were abundant in some regions of the cerebellum. Life span was lengthened in NPC mice with the implanted NSCs. However, the rate of weight gain and subsequent weight loss, resulting from neurodegeneration, was not significantly different from un-injected controls. Ataxia was measured by Rota-Rod performance. The overall rate of decline in time on the Rota-Rod was not significantly slowed down. Thus, in this small group of NPC mice, a single administration in the neonatal period of the NSCs (which were not engineered to over-express the missing gene and not directed into the parenchyma) was only partially therapeutic.

Neural Progenitor Cell Transplantation and Imaging in a Large Animal Model

Neuroscience Research. Nov, 2007  |  Pubmed ID: 17897743

To evaluate neural stem/progenitor cell (NPC) transplantation therapy in cat models of neurodegenerative diseases, we have isolated, expanded and characterized feline NPCs (fNPCs) from normal fetal cat brain. Feline NPCs responsive to both human epidermal growth factor (hEGF) and human fibroblast growth factor 2 (hFGF2) proliferated as neurospheres, which were able to differentiate to neurons and glial cells. The analysis of growth factors indicated that both hEGF and hFGF2 were required for proliferation of fNPCs. In contrast to the effect on human NPCs, human leukemia inhibitory factor (hLIF) enhanced differentiation of fNPCs. Expanded fNPCs were injected into the brains of normal adult cats. Immunohistochemical analysis showed that the majority of transplanted cells were located adjacent to the injection site and some fNPCs differentiated into neurons. The survival of transplanted fNPCs over time was monitored using non-invasive bioluminescent imaging technology. This study provided the first evidence of allotransplantation of fNPCs into feline CNS. Cats have heterogeneous genetic backgrounds and possess neurological diseases that closely resemble analogous human diseases. The characterization of fNPCs and exploration of non-invasive bioluminescent imaging to track transplanted cells in this study will allow evaluation of NPC transplantation therapy using feline models of human neurological diseases.

Injection of Mouse and Human Neural Stem Cells into Neonatal Niemann-Pick A Model Mice

Brain Research. Apr, 2007  |  Pubmed ID: 17289003

Cloned mouse C17.2 neural stem cells (NSCs) or human NSCs were injected into five CNS sites in very large numbers (100,000 cells/site, or a total of 500,000 cells) into 18 neonatal mice homozygous for a targeted deletion (knockout) of the acid sphingomyelinase (ASM) gene (called ASMKO mice), a faithful model of human Niemann-Pick type A (NP-A) disease, and into 10 wild-type mice, all on the C57BL/6J background. Injected mice were not immunosuppressed, and all survived to adulthood. Non-injected ASMKO controls had developed widespread neuronal and glial vacuolation and lysosomal accumulation of sphingomyelin and cholesterol when examined histologically at 16 weeks of age. Unlike children with NP-A disease, the ASMKO mice also lose cerebellar Purkinje neurons progressively, are ataxic, and show parallel progressive declines in rotorod performance. At 16 weeks NSC-injected mice showed a dramatic decrease in neuronal and glial vacuolation (by standard histological staining) and in cholesterol accumulation (by filipin fluorescence staining) throughout the cerebral neocortex, hippocampal formation, striatum and cerebellum, with lesser but clear improvement throughout the brainstem. Improvement was modestly but consistently better in human HFT13-injected than in mouse C17.2-injected ASMKO mice. Improvement in the ASMKO brains was more widespread than the distribution of NSCs, an indication that ASM must have been secreted and diffused at therapeutic concentrations beyond the territory occupied by NSCs. However, though Purkinje cell rescue has been achieved with NSCs in some other disease models, loss of Purkinje neurons and decline in rotorod performance were still present in injected ASMKO mice.

Stem Cells Act Through Multiple Mechanisms to Benefit Mice with Neurodegenerative Metabolic Disease

Nature Medicine. Apr, 2007  |  Pubmed ID: 17351625

Intracranial transplantation of neural stem cells (NSCs) delayed disease onset, preserved motor function, reduced pathology and prolonged survival in a mouse model of Sandhoff disease, a lethal gangliosidosis. Although donor-derived neurons were electrophysiologically active within chimeric regions, the small degree of neuronal replacement alone could not account for the improvement. NSCs also increased brain beta-hexosaminidase levels, reduced ganglioside storage and diminished activated microgliosis. Additionally, when oral glycosphingolipid biosynthesis inhibitors (beta-hexosaminidase substrate inhibitors) were combined with NSC transplantation, substantial synergy resulted. Efficacy extended to human NSCs, both to those isolated directly from the central nervous system (CNS) and to those derived secondarily from embryonic stem cells. Appreciating that NSCs exhibit a broad repertoire of potentially therapeutic actions, of which neuronal replacement is but one, may help in formulating rational multimodal strategies for the treatment of neurodegenerative diseases.

Isolation of Amniotic Stem Cell Lines with Potential for Therapy

Nature Biotechnology. Jan, 2007  |  Pubmed ID: 17206138

Stem cells capable of differentiating to multiple lineages may be valuable for therapy. We report the isolation of human and rodent amniotic fluid-derived stem (AFS) cells that express embryonic and adult stem cell markers. Undifferentiated AFS cells expand extensively without feeders, double in 36 h and are not tumorigenic. Lines maintained for over 250 population doublings retained long telomeres and a normal karyotype. AFS cells are broadly multipotent. Clonal human lines verified by retroviral marking were induced to differentiate into cell types representing each embryonic germ layer, including cells of adipogenic, osteogenic, myogenic, endothelial, neuronal and hepatic lineages. Examples of differentiated cells derived from human AFS cells and displaying specialized functions include neuronal lineage cells secreting the neurotransmitter L-glutamate or expressing G-protein-gated inwardly rectifying potassium channels, hepatic lineage cells producing urea, and osteogenic lineage cells forming tissue-engineered bone.

Genetic Modification of Neural Stem Cells

Molecular Therapy : the Journal of the American Society of Gene Therapy. Mar, 2008  |  Pubmed ID: 18253153

Neural stem cells (NSCs) are the main vehicle for genetic and molecular therapies in the central nervous system (CNS). The sustainability of NSCs has been ensured through genetic manipulation both in vitro and in vivo. NSC lines have also been immortalized and controlled for cell growth in similar fashion. Their potential to differentiate and their genetic plasticity make them the modality of choice for cellular transplantation. After transplantation, NSCs also exhibit inherent long-distance migratory capabilities and a remarkable capacity to integrate into brain structures. This makes NSCs the ideal candidate for delivery and expression of therapeutic genes. Mouse models of CNS diseases have already demonstrated the efficacy of such NSC-mediated treatment, and further investigations are underway to bridge the gap into true clinical application. Finally, the imaging possibilities with NSC transplants are endless, and they will be a pivotal component to safe and effective human transplantation. This paper provides an overview on NSCs and the various methods in which they have been genetically manipulated for biological investigation.

Stem Cell-mediated Regeneration of the Intervertebral Disc: Cellular and Molecular Challenge

Neurosurgical Focus. 2008  |  Pubmed ID: 18341398

Regenerative medicine and stem cells hold great promise for intervertebral disc (IVD) disease. The therapeutic implications of utilizing stem cells to repair degenerated discs and treat back pain are highly anticipated by both the clinical and scientific communities. Although the avascular environment of the IVD poses a challenge for stem cell-mediated regeneration, neuroprogenitor cells have been discovered within degenerated discs, allowing scientists to revisit the hostile environment of the IVD as a target for stem cell therapy. Issues now under investigation include the timing of cell delivery and manipulation of stem cells to make them more efficient and adaptive in the IVD niche. This review covers the mechanisms underlying disc degeneration as well as the molecular and cellular challenges involved in directing stem cells to the desired cell type for intradiscal transplantation.

Brain Tumor Stem Cells and the Tumor Microenvironmen

Neurosurgical Focus. 2008  |  Pubmed ID: 18341404

Recent advances in stem cell research and developmental neurobiology have uncovered new perspectives from which to investigate various forms of cancer. Specifically, the hypothesis that tumors consist of a subpopulation of malignant cells similar to stem cells is of great interest to scientists and clinicians and has been dubbed the "cancer stem cell hypothesis." The region in which this assertion is most relevant is within the brain. Cancer stem cells have been isolated from brain tumors that exhibit characteristics of differentiation and proliferation normally seen only in neural stem cells. These cancer stem cells may be responsible for tumor origin, survival, and proliferation. Furthermore, these cells must be considered within their immediate microenvironment when investigating mechanisms of tumorigenesis. Evidence of brain tumor stem cells is reviewed along with the role of tumor environment as the context within which these cells should be understood.

Human Neural Stem Cells Migrate Along the Nigrostriatal Pathway in a Primate Model of Parkinson's Disease

Experimental Neurology. Jun, 2008  |  Pubmed ID: 18394605

Although evidence of damage-directed neural stem cell (NSC) migration has been well-documented in the rodent, to our knowledge it has never been confirmed or quantified using human NSC (hNSC) in an adult non-human primate modeling a human neurodegenerative disease state. In this report, we attempt to provide that confirmation, potentially advancing basic stem cell concepts toward clinical relevance. hNSCs were implanted into the caudate nucleus (bilaterally) and substantia nigra (unilaterally) of 7, adult St. Kitts African green monkeys (Chlorocebus sabaeus) with previous exposure to systemic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin that disrupts the dopaminergic nigrostriatal pathway. A detailed quantitative analysis of hNSC migration patterns at two time points (4 and 7 months) following transplantation was performed. Density contour mapping of hNSCs along the dorsal-ventral and medial-lateral axes of the brain suggested that >80% of hNSCs migrated from the point of implantation to and along the impaired nigrostriatal pathway. Although 2/3 of hNSCs were transplanted within the caudate, <1% of 3x10(6) total injected donor cells were identified at this site. The migrating hNSC did not appear to be pursuing a neuronal lineage. In the striatum and nigrostriatal pathway, but not in the substantia nigra, some hNSCs were found to have taken a glial lineage. The property of neural stem cells to align themselves along a neural pathway rendered dysfunctional by a given disease is potentially a valuable clinical tool.

Neural Stem Cell Transplantation in Mouse Brain

Current Protocols in Neuroscience / Editorial Board, Jacqueline N. Crawley ... [et Al.]. Jan, 2008  |  Pubmed ID: 18428671

Neural stem cells (NSCs) are the most primordial, least committed cells of the nervous system, and transplantation of these multipotent cells holds the promise of regenerative therapy for many central nervous system (CNS) diseases. This unit describes methods for NSC transplantation into neonatal mouse pups, embryonic mouse brain, and adult mouse brain. A description of options for detection of labeled donor cells in engrafted mouse brain is provided along with an example protocol for detecting lacZ-expressing cells in situ. Also included is a protocol for preparing NSCs for transplantation.

Inflammation As a Matchmaker: Revisiting Cell Fusion

Nature Cell Biology. May, 2008  |  Pubmed ID: 18454127

Neural Stem Cell Delivery to the Spinal Cord in an Ovine Model of Fetal Surgery for Spina Bifida

Surgery. Sep, 2008  |  Pubmed ID: 18707035

We introduce the notion of prenatal neural stem cell (NSC) delivery to the spinal cord as an adjuvant to fetal repair of spina bifida.

Preparation of Autogenic Human Feeder Cells for Growth of Human Embryonic Stem Cells

Current Protocols in Stem Cell Biology. Mar, 2008  |  Pubmed ID: 18770638

Human embryonic stem cells (hESCs) that are currently distributed under NIH guidelines, as well as many of those that are not on the NIH registry, have been derived and maintained in coculture with growth-arrested mouse embryonic fibroblasts (MEFs). Using this mouse support system may compromise the therapeutic potential of these hESCs because of the risk of transmitting xenopathogens. Alternatively, to reduce this risk, methods to culture undifferentiated hESCs on autologous hESC-derived human feeder layers have now been developed. This feeder cell system derived from hESCs successfully prolongs growth of undifferentiated hESCs and eliminates risk factors and concerns about using xenogeneic or unknown allogeneic feeders. In this unit, we provide the necessary protocols for an autogeneic human feeder system that efficiently supports hESC growth and maintenance of pluripotency. Curr. Protoc. Stem Cell Biol. 4:1C.5.1-1C.5.15. (c) 2008 by John Wiley & Sons, Inc.

Neural Stem/progenitor Cells Modulate Immune Responses by Suppressing T Lymphocytes with Nitric Oxide and Prostaglandin E2

Experimental Neurology. Mar, 2009  |  Pubmed ID: 19109951

We and others have reported that neural stem/progenitor cells (NSCs) may exert direct anti-inflammatory activity. This action has been attributed, in part, to T-cell suppression. However, how T-cells become suppressed by NSCs remains unresolved. In this study, we explored one of these mechanisms and challenged some previously advanced hypotheses regarding underlying NSC-mediated T-cell suppression. We employed an easily observable and manipulatable system in which activated and non-activated T-cells were co-cultured with a stable well-characterized clone of lacZ-expressing murine NSCs. As in previous reports, NSCs were found to inhibit T-cell proliferation. However, this inhibition by NSCs was not due to suppression of T cell activation or induction of apoptosis of T cells during the early activation stage. High levels of nitric oxide (NO) and prostaglandin E2 (PGE2) were induced in the T cells when co-cultured with NSCs. In addition, inducible NOS (iNOS) and microsomal type 1 PGES (mPGES-1) were readily detected in NSCs in co-culture with T-cells, but not at all in NSCs cultured alone or in activated T cells cultured with or without NSCs. This finding suggested that activated T cells induced NO and PGE2 production in the NSCs. Furthermore, T-cell proliferation inhibited by co-culture with the NSCs was significantly restored by inhibitors of NO and PGE2 production. Therefore, NSCs appear to suppress T-cells, at least in part, by NO and PGE2 production which, in turn, would account for the well-documented reduction of central nervous system immunopathology by transplanted NSCs.

Neural Precursor Cell Lines Promote Neurite Branching

The International Journal of Neuroscience. 2009  |  Pubmed ID: 19116829

Schwann cells and primary progenitor cells improve regeneration across peripheral nerve defects. This study examined the impact of immortalized neural precursor cells on regeneration of rat nerve defects. Across 10-mm gaps, neuromas formed without neural cables with C17.2- or RN33B-transplanted cells, but neural cables formed across 5-mm gaps seeded with RN33B cells. In vitro, dorsal root ganglia neurites elongated across Schwann and RN33B cells; RN33B cells induced neurite branching with shorter total outgrowth. Neural cable formation in vivo was likely determined by the balance of guidance and branch-inducing factors secreted by Schwann and transplanted precursor cells.

Role of Monocyte Chemoattractant Protein-1 (MCP-1/CCL2) in Migration of Neural Progenitor Cells Toward Glial Tumors

Journal of Neuroscience Research. May, 2009  |  Pubmed ID: 19125409

Neural progenitor cells (NPCs) have been investigated as potential vehicles for brain tumor therapy because they have been shown to migrate toward central nervous system gliomas and can be genetically engineered to deliver cytotoxic agents to tumors. The mechanisms that regulate migration of NPCs to tumors are not fully understood. By means of microarray analysis, polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemistry, we found that monocyte chemoattractant protein-1 (MCP-1/CCL-2) was expressed in experimental brain tumor cells in vivo and in vitro. CCR2, the receptor for MCP-1, was expressed on C17.2 NPCs. We used a modified Boyden chamber assay and found increased migration of NPCs in vitro in response to MCP-1. By means of an in vivo model for NPC migration, we found evidence of NPC migration toward areas of MCP-1 infusion in rat brains. An understanding of NPC migration mechanisms may be used to enhance delivery of cytotoxic agents to brain tumor cells.

Discovery of a Functional Protein Complex of Netrin-4, Laminin Gamma1 Chain, and Integrin Alpha6beta1 in Mouse Neural Stem Cells

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2009  |  Pubmed ID: 19193855

Molecular and cellular interactions coordinating the origin and fate of neural stem cells (NSCs) in the adult brain are far from being understood. We present a protein complex that controls proliferation and migration of adult NSCs destined for the mouse olfactory bulb (OB). Combinatorial selection based on phage display technology revealed a previously unrecognized complex between the soluble protein netrin-4 and laminin gamma1 subunit that in turn activates an alpha6beta1 integrin-mediated signaling pathway in NSCs. Differentiation of NSCs is accompanied by a decrease in netrin-4 receptors, indicating that netrin-4 participates in the continual propagation of this stem cell population. Notably, the stem cells themselves do not synthesize netrin-4. Further, we show that netrin-4 is produced by selected GFAP-positive astrocytes positioned close to newborn neurons migrating in the anterior part of the rostral migratory stream (RMS) and within the OB. Our findings present a unique molecular mechanism mediating astrocytic/neuronal crosstalk that regulates ongoing neurogenesis in the adult olfactory system.

Neuroimaging As a Basis for Rational Stem Cell Therapy

Pediatric Neurology. Mar, 2009  |  Pubmed ID: 19218036

Neonatal global or focal hypoxic-ischemic brain injury remains a frequent and devastating condition, with serious long-term sequelae. An important issue in any neonatal clinical trial of neuroprotective agents relates to developing accurate measures of injury severity and also suitable measures of the response to treatment. Advanced magnetic resonance imaging techniques can acquire serial and noninvasive data about brain structure, metabolic activity, and the response to injury or treatment. These imaging methods need validation in appropriate animal models for translational research studies in human newborns. This review describes several approaches that use imaging as well as proton magnetic resonance spectroscopy to assess the severity of ischemic injury (e.g., for possible candidate selection) and for monitoring the progression and evolution of injury over time and as an indicator of recovery or response to treatment. Preliminary data are presented on how imaging can be used after neural stem cell implantation to characterize the migration rate, the magnitude of stem cell proliferation, and their final location. Imaging has the potential to allow monitoring of many dimensions of neuroprotective treatments and can be expected to contribute to efficacy and safety when clinical trials using neural stem cells or other neuroprotective agents become available.

The Dynamics of Long-term Transgene Expression in Engrafted Neural Stem Cells

The Journal of Comparative Neurology. Jul, 2009  |  Pubmed ID: 19399895

To assess the dynamics and confounding variables that influence transgene expression in neural stem cells (NSCs), we generated distinct NSC clones from the same pool of cells, carrying the same reporter gene transcribed from the same promoter, transduced by the same retroviral vector, and transplanted similarly at the same differentiation state, at the same time and location, into the brains of newborn mouse littermates, and monitored in parallel for over a year in vivo (without immunosuppression). Therefore, the sole variables were transgene chromosomal insertion site and copy number. We then adapted and optimized a technique that tests, at the single cell level, persistence of stem cell-mediated transgene expression in vivo based on correlating the presence of the transgene in a given NSC's nucleus (by fluorescence in situ hybridization [FISH]) with the frequency of that transgene's product within the same cell (by combined immunohistochemistry [IHC]). Under the above-stated conditions, insertion site is likely the most contributory variable dictating transgene downregulation in an NSC after 3 months in vivo. We also observed that this obstacle could be effectively and safely counteracted by simple serial infections (as few as three) inserting redundant copies of the transgene into the prospective donor NSC. (The preservation of normal growth control mechanisms and an absence of tumorigenic potential can be readily screened and ensured ex vivo prior to transplantation.) The combined FISH/IHC strategy employed here for monitoring the dynamics of transgene expression at the single cell level in vivo may be used for other types of therapeutic and housekeeping genes in endogenous and exogenous stem cells of many organs and lineages.

Long-term Multilayer Adherent Network (MAN) Expansion, Maintenance, and Characterization, Chemical and Genetic Manipulation, and Transplantation of Human Fetal Forebrain Neural Stem Cells

Current Protocols in Stem Cell Biology. May, 2009  |  Pubmed ID: 19455542

Human neural stem/precursor cells (hNSC/hNPC) have been targeted for application in a variety of research models and as prospective candidates for cell-based therapeutic modalities in central nervous system (CNS) disorders. To this end, the successful derivation, expansion, and sustained maintenance of undifferentiated hNSC/hNPC in vitro, as artificial expandable neurogenic micro-niches, promises a diversity of applications as well as future potential for a variety of experimental paradigms modeling early human neurogenesis, neuronal migration, and neurogenetic disorders, and could also serve as a platform for small-molecule drug screening in the CNS. Furthermore, hNPC transplants provide an alternative substrate for cellular regeneration and restoration of damaged tissue in neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease. Human somatic neural stem/progenitor cells (NSC/NPC) have been derived from a variety of cadaveric sources and proven engraftable in a cytoarchitecturally appropriate manner into the developing and adult rodent and monkey brain while maintaining both functional and migratory capabilities in pathological models of disease. In the following unit, we describe a new procedure that we have successfully employed to maintain operationally defined human somatic NSC/NPC from developing fetal, pre-term post-natal, and adult cadaveric forebrain. Specifically, we outline the detailed methodology for in vitro expansion, long-term maintenance, manipulation, and transplantation of these multipotent precursors.

Maintaining and Engineering Neural Stem Cells for Delivery of Genetically Encoded Therapy to Brain Tumors

Methods in Molecular Biology (Clifton, N.J.). 2009  |  Pubmed ID: 19582432

Despite advances for the treatment of cancer, the prognosis for patients suffering from malignant brain tumors remains dismal. High-grade neoplasms, such as gliomas, are highly invasive and spawn widely disseminated microsatellites that have limited the efficacy of surgical and adjunctive therapies. The cancer stem cell hypothesis suggests that conventional chemotherapeutic treatments kill differentiated and differentiating cells which often form the bulk of the tumor. One major concern is that the cells which give rise to the tumor, the cancer stem cells, remain untouched and may be responsible for a relapse of the disease. Therefore, an adjunctive therapy to current cancer treatment is critical for the survivability of patients suffering from brain tumors. We have successfully engineered tumor-tropic neural stem cells to deliver antineoplastic gene products directly to the tumor-producing cells. This potential therapeutic strategy may safely eradicate tumor-producing cells in the brain while minimizing damage to normal, healthy cells.

Neural Stem Cell Transplantation Benefits a Monogenic Neurometabolic Disorder During the Symptomatic Phase of Disease

Stem Cells (Dayton, Ohio). Sep, 2009  |  Pubmed ID: 19591217

Although we and others have demonstrated that neural stem cells (NSCs) may impact such neurogenetic conditions as lysosomal storage diseases when transplanted at birth, it has remained unclear whether such interventions can impact well-established mid-stage disease, a situation often encountered clinically. Here we report that when NSCs were injected intracranially into the brain of adult symptomatic Sandhoff (Hexb(-/-)) mice, cells migrated far from the injection site and integrated into the host cytoarchitecture, restoring beta-hexosaminidase enzyme activity and promoting neuropathologic and behavioral improvement. Mouse lifespan increased, neurological function improved, and disease progression was slowed. These clinical benefits correlated with neuropathological correction at the cellular and molecular levels, reflecting the multiple potential beneficial actions of stem cells, including enzyme cross-correction, cell replacement, tropic support, and direct anti-inflammatory action. Pathotropism (i.e., migration and homing of NSCs to pathological sites) could be imaged in real time by magnetic resonance imaging. Differentially expressed chemokines might play a role in directing the migration of transplanted stem cells to sites of pathology. Significantly, the therapeutic impact of NSCs implanted in even a single location was surprisingly widespread due to both cell migration and enzyme diffusion. Because many of the beneficial actions of NSCs observed in newborn brains were recapitulated in adult brains to the benefit of Sandhoff recipients, NSC-based interventions may also be useful in symptomatic subjects with established disease.

Phosphoproteomic Analysis of Human Embryonic Stem Cells

Cell Stem Cell. Aug, 2009  |  Pubmed ID: 19664994

Protein phosphorylation, while critical to cellular behavior, has been undercharacterized in pluripotent cells. Therefore, we performed phosphoproteomic analyses of human embryonic stem cells (hESCs) and their differentiated derivatives. A total of 2546 phosphorylation sites were identified on 1602 phosphoproteins; 389 proteins contained more phosphorylation site identifications in undifferentiated hESCs, whereas 540 contained more such identifications in differentiated derivatives. Phosphoproteins in receptor tyrosine kinase (RTK) signaling pathways were numerous in undifferentiated hESCs. Cellular assays corroborated this observation by showing that multiple RTKs cooperatively supported undifferentiated hESCs. In addition to bFGF, EGFR, VEGFR, and PDGFR activation was critical to the undifferentiated state of hESCs. PDGF-AA complemented a subthreshold bFGF concentration to maintain undifferentiated hESCs. Also consistent with phosphoproteomics, JNK activity participated in maintenance of undifferentiated hESCs. These results support the utility of phosphoproteomic data, provide guidance for investigating protein function in hESCs, and complement transcriptomics/epigenetics for broadening our understanding of hESC fate determination.

Culture System for Rodent and Human Oligodendrocyte Specification, Lineage Progression, and Maturation

Current Protocols in Stem Cell Biology. Sep, 2009  |  Pubmed ID: 19725014

Here we document protocols for the production, isolation, and maintenance of the oligodendrocyte phenotype from rodent and human neural stem cells. Our unique method relies on a series of chemically defined media, specifically designed and carefully characterized for each developmental stage of oligodendrocytes as they advance from oligodendrocyte progenitors to mature, myelinating oligodendrocytes.

A Safer Stem Cell: on Guard Against Cancer

Nature Medicine. Sep, 2009  |  Pubmed ID: 19734873

Cross-talk Between Stem Cells and the Dysfunctional Brain is Facilitated by Manipulating the Niche: Evidence from an Adhesion Molecule

Stem Cells (Dayton, Ohio). Nov, 2009  |  Pubmed ID: 19785036

In the injured brain, the behavior of neural stem/progenitor cells (NSCs) is regulated by multiple converging factors encountered in the niche, which is composed of several neural and non-neural cell types. Signals emanating from the host influence the migration, survival, distribution, and fate of transplanted NSCs, which in turn can create host microenvironments that favor a return to homeostasis. We tested the hypothesis that overexpression of key facilitatory molecules that define the injury niche might enhance this bidirectional stem cell-host interaction to therapeutic advantage. As proof of concept, we investigated whether conditioning the niche with the neural cell adhesion molecule L1 might enhance recovery in a prototypical neurodegenerative milieu-the MPTP-induced model of Parkinson's disease in aged mice-where cross-talk between NSCs and imperiled host dopaminergic neurons is known to be pivotal in rescuing the function and connectivity of the latter. In lesioned mice (and in unlesioned controls), we overexpressed L1 in the NSCs to be transplanted into the ventral mesencephalon. Several pairwise experimental combinations were tested based on variations of engrafting L1 overexpressing versus nonoverexpressing NSCs into wild-type (WT) versus L1-overexpressing transgenic mice (specifically L1 transcribed from the GFAP promoter and, hence, overexpressed in host astrocytes). Enrichment for L1-particularly when expressed simultaneously in both donor NSCs and host brain-led to rapid and extensive distribution of exogenous NSCs, which in turn rescued (with an efficacy greater than in nonengineered controls) dysfunctional host dopaminergic nigral neurons, even when grafting was delayed by a month. L1 overexpression by NSCs also enhanced their own differentiation into tyrosine hydroxylase-expressing neurons in both WT and transgenic hosts. Graft-host interactions were thus favored by progressively increasing levels of L1. More broadly, this study supports the view that manipulating components of the niche (such as an adhesion molecule) that facilitate cross-talk between stem cells and the dysfunctional brain may offer new strategies for more efficacious neurotransplantation, particularly when treatment is delayed as in chronic lesions or advanced stages of a neurodegenerative disease.

Important Precautions when Deriving Patient-specific Neural Elements from Pluripotent Cells

Cytotherapy. 2009  |  Pubmed ID: 19903095

Multipotent human neural stem cells (hNSC) have traditionally been isolated directly from the central nervous system (CNS). To date, as a therapeutic tool in the treatment of neurologic disorders, the most promising results have been obtained using hNSC isolated directly from the human fetal neuroectoderm. The propagation ability of such tissue-derived hNSC is often limited, however, making it difficult to establish a large-scale culture. Following engraftment, these hNSC often show low efficiency in generating the desired neuronal cells necessary for reconstruction of the damaged host milieu and, as a result, have failed to give satisfactory results in clinical trials so far. Alternatively, human embryonic stem cells (hESC) offer a pluripotent reservoir for in vitro derivation of a rich spectrum of well-characterized neural-lineage committed stem/progenitor/precursor cells that can, theoretically, be picked at precisely their safest and most efficacious state of plasticity to meet a given clinical challenge. However, the need for 'foreign' biologic additives and multilineage differentiation inclination may make direct use of such cell-derived hNSC in patients problematic. The hNSC, when derived from pluripotent cells under protocols presently employed in the field, tend to display not only a low efficiency in neuronal differentiation, but also an inclination for phenotypic heterogeneity and instability and, hence, increased risk of tumorigenesis following engraftment. For hNSC derived in vitro to be used safely in therapeutic paradigms, it requires conversion of human pluripotent cells uniformly to cells that are restricted to the neural lineage in need of repair. Developing strategies for direct induction of human pluripotent cells exclusively into neural-committed progenies at a broad range of developmental stages will allow a large supply of optimal therapeutic hNSC tailor-made for safe and effective treatment of particular neurologic diseases and injuries in patients.

The Transcriptional Network for Mesenchymal Transformation of Brain Tumours

Nature. Jan, 2010  |  Pubmed ID: 20032975

The inference of transcriptional networks that regulate transitions into physiological or pathological cellular states remains a central challenge in systems biology. A mesenchymal phenotype is the hallmark of tumour aggressiveness in human malignant glioma, but the regulatory programs responsible for implementing the associated molecular signature are largely unknown. Here we show that reverse-engineering and an unbiased interrogation of a glioma-specific regulatory network reveal the transcriptional module that activates expression of mesenchymal genes in malignant glioma. Two transcription factors (C/EBPbeta and STAT3) emerge as synergistic initiators and master regulators of mesenchymal transformation. Ectopic co-expression of C/EBPbeta and STAT3 reprograms neural stem cells along the aberrant mesenchymal lineage, whereas elimination of the two factors in glioma cells leads to collapse of the mesenchymal signature and reduces tumour aggressiveness. In human glioma, expression of C/EBPbeta and STAT3 correlates with mesenchymal differentiation and predicts poor clinical outcome. These results show that the activation of a small regulatory module is necessary and sufficient to initiate and maintain an aberrant phenotypic state in cancer cells.

Screening the Mammalian Extracellular Proteome for Regulators of Embryonic Human Stem Cell Pluripotency

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2010  |  Pubmed ID: 20133595

Approximately 3,500 mammalian genes are predicted to be secreted or single-pass transmembrane proteins. The function of the majority of these genes is still unknown, and a number of the encoded proteins might find use as new therapeutic agents themselves or as targets for small molecule or antibody drug development. To analyze the physiological activities of the extracellular proteome, we developed a large-scale, high-throughput protein expression, purification, and screening platform. For this study, the complete human extracellular proteome was analyzed and prioritized based on genome-wide disease association studies to select 529 initial target genes. These genes were cloned into three expression vectors as native sequences and as N-terminal and C-terminal Fc fusions to create an initial collection of 806 purified secreted proteins. To determine its utility, this library was screened in an OCT4-based cellular assay to identify regulators of human embryonic stem-cell self-renewal. We found that the pigment epithelium-derived factor can promote long-term pluripotent growth of human embryonic stem cells without bFGF or TGFbeta/Activin/Nodal ligand supplementation. Our results further indicate that activation of the pigment epithelium-derived factor receptor-Erk1/2 signaling pathway by the pigment epithelium-derived factor is sufficient to maintain the self-renewal of pluripotent human embryonic stem cells. These experiments illustrate the potential for discovering novel biological functions by directly screening protein diversity in cell-based phenotypic or reporter assays.

Communication Via Gap Junctions Underlies Early Functional and Beneficial Interactions Between Grafted Neural Stem Cells and the Host

Proceedings of the National Academy of Sciences of the United States of America. Mar, 2010  |  Pubmed ID: 20147621

How grafted neural stem cells (NSCs) and their progeny integrate into recipient brain tissue and functionally interact with host cells is as yet unanswered. We report that, in organotypic slice cultures analyzed by ratiometric time-lapse calcium imaging, current-clamp recordings, and dye-coupling methods, an early and essential way in which grafted murine or human NSCs integrate functionally into host neural circuitry and affect host cells is via gap-junctional coupling, even before electrophysiologically mature neuronal differentiation. The gap junctions, which are established rapidly, permit exogenous NSCs to influence directly host network activity, including synchronized calcium transients with host cells in fluctuating networks. The exogenous NSCs also protect host neurons from death and reduce such signs of secondary injury as reactive astrogliosis. To determine whether gap junctions between NSCs and host cells may also mediate neuroprotection in vivo, we examined NSC transplantation in two murine models characterized by degeneration of the same cell type (Purkinje neurons) from different etiologies, namely, the nervous and SCA1 mutants. In both, gap junctions (containing connexin 43) formed between NSCs and host cells at risk, and were associated with rescue of neurons and behavior (when implantation was performed before overt neuron loss). Both in vitro and in vivo beneficial NSC effects were abrogated when gap junction formation or function was suppressed by pharmacologic and/or RNA-inhibition strategies, supporting the pivotal mediation by gap-junctional coupling of some modulatory, homeostatic, and protective actions on host systems as well as establishing a template for the subsequent development of electrochemical synaptic intercellular communication.

Derivation of Vasculature from Embryonic Stem Cells

Current Protocols in Stem Cell Biology. Mar, 2010  |  Pubmed ID: 20200855

The formation of the multicellular vascular system is critical to the growth, development, and viability of an organism, and many embryonic lethal mouse knockouts are due to vascular defects. Unfortunately, the complex nature, and many cell types involved in vasculogenesis and angiogenesis has stymied in vitro models of vascular formation. This unit describes a system that allows human embryonic stem cells to differentiate and spontaneously form vascular networks via both vasculogenesis and angiogenesis in the context of the three germ layers.

Microarray-based Transcriptional and Epigenetic Profiling of Matrix Metalloproteinases, Collagens, and Related Genes in Cancer

The Journal of Biological Chemistry. Jun, 2010  |  Pubmed ID: 20404328

Epigenetic parameters (DNA methylation, histone modifications, and miRNAs) play a significant role in cancer. To identify the common epigenetic signatures of both the individual matrix metalloproteinases (MMPs) and the additional genes, the function of which is also linked to proteolysis, migration, and tumorigenesis, we performed epigenetic profiling of 486 selected genes in unrelated non-migratory MCF-7 breast carcinoma and highly migratory U251 glioma cells. Genome-wide transcriptional profiling, quantitative reverse transcription-PCR, and microRNA analyses were used to support the results of our epigenetic studies. Transcriptional silencing in both glioma and breast carcinoma cells predominantly involved the repressive histone H3 Lys-27 trimethylation (H3K27me3) mark. In turn, epigenetic stimulation was primarily performed through a gain in the histone H3 Lys-4 dimethylation (H3K4me2) and H3 hyperacetylation and by a global reduction of H3K27me3. Inactive pro-invasive genes in MCF-7 cells but not in U251 cells frequently exhibited a stem cell-like bivalent mark (enrichment in both H3K27me3 and H3K4me2), a characteristic of developmental genes. In contrast with other MMPs, MMP-8 was epigenetically silenced in both cell types, thus providing evidence for the strict epigenetic control of this anti-tumorigenic proteinase in cancer. Epigenetic stimulation of multiple collagen genes observed in cultured glioma cells was then directly confirmed using orthotopic xenografts and tumor specimens. We suggest that the epigenetic mechanisms allow gliomas to deposit an invasion-promoting collagen-enriched matrix and then to use this matrix to accomplish their rapid migration through the brain tissue.

Culture and Manipulation of Neural Stem Cells

Advances in Experimental Medicine and Biology. 2010  |  Pubmed ID: 20455492

Despite advances in the treatment of cancer, the prognosis of patient diagnosed with metastatis cancer to the brain remains poor. The role of neural stem cells as a viable tool in the treatment of metastatic cancer to the brain alone or in conjuction with current therapeutic modalities is promising. Both murine and human neural stem cells (NSCs) have been shown to migrate through the central nervous system (CNS) and infiltrate tumors and other pathological disease states of the brain. Genetic modification of NSCs to produce cytotoxic or immunomodulatory agents in the vicinity of a primary tumor and/or satellite lesion or has proven instrumental to the reduction of tumor bulk in murine models. Although the use of stem cells proves to be a volatile social topic, scientists have discovered that NSCs are present in the adult brain and continue to propagate and differentiate. These cells may be isolated and cultured to produce clonal NSC lines that are capable of self renewal and differentiation when introduced into the CNS. In this chapter, we describe protocols currently used in ourlab for the successful maintenance of NSCs in vitro advancing the role of neural stem cells in the treatment of brain tumors.

Nna1 Mediates Purkinje Cell Dendritic Development Via Lysyl Oxidase Propeptide and NF-κB Signaling

Neuron. Oct, 2010  |  Pubmed ID: 20920790

The molecular pathways controlling cerebellar Purkinje cell dendrite formation and maturation are poorly understood. The Purkinje cell degeneration (pcd) mutant mouse is characterized by mutations in Nna1, a gene discovered in an axonal regenerative context, but whose actual function in development and disease is unknown. We found abnormal development of Purkinje cell dendrites in postnatal pcd(Sid) mice and linked this deficit to a deletion mutation in exon 7 of Nna1. With single cell gene profiling and virus-based gene transfer, we analyzed a molecular pathway downstream to Nna1 underlying abnormal Purkinje cell dendritogenesis in pcd(Sid) mice. We discovered that mutant Nna1 dramatically increases intranuclear localization of lysyl oxidase propeptide, which interferes with NF-κB RelA signaling and microtubule-associated protein regulation of microtubule stability, leading to underdevelopment of Purkinje cell dendrites. These findings provide insight into Nna1's role in neuronal development and why its absence renders Purkinje cells more vulnerable.

Early Acquisition of Neural Crest Competence During HESCs Neuralization

PloS One. 2010  |  Pubmed ID: 21085480

Neural crest stem cells (NCSCs) are a transient multipotent embryonic cell population that represents a defining characteristic of vertebrates. The neural crest (NC) gives rise to many derivatives including the neurons and glia of the sensory and autonomic ganglia of the peripheral nervous system, enteric neurons and glia, melanocytes, and the cartilaginous, bony and connective tissue of the craniofacial skeleton, cephalic neuroendocrine organs, and some heart vessels.

Neurotransplantation of Stem Cells Genetically Modified to Express Human Dopamine Transporter Reduces Alcohol Consumption

Stem Cell Research & Therapy. 2010  |  Pubmed ID: 21122109

Regulated neurotransmitter actions in the mammalian central nervous system determine brain function and control peripheral organs and behavior. Although drug-seeking behaviors, including alcohol consumption, depend on central neurotransmission, modification of neurotransmitter actions in specific brain nuclei remains challenging. Herein, we report a novel approach for neurotransmission modification in vivo by transplantation of stem cells engineered to take up the neurotransmitter dopamine (DA) efficiently through the action of the human dopamine transporter (hDAT). As a functional test in mice, we used voluntary alcohol consumption, which is known to release DA in nucleus accumbens (NAC), an event hypothesized to help maintain drug-seeking behavior. We reasoned that reducing extracellular DA levels, by engrafting into NAC DA-sequestering stem cells expressing hDAT, would alter alcohol intake.

Self-renewal Induced Efficiently, Safely, and Effective Therapeutically with One Regulatable Gene in a Human Somatic Progenitor Cell

Proceedings of the National Academy of Sciences of the United States of America. Mar, 2011  |  Pubmed ID: 21378266

In the field of induced potency and fate reprogramming, it remains unclear what the best starting cell might be and to what extent a cell need be transported back to a more primitive state for translational purposes. Reprogramming a committed cell back to pluripotence to then instruct it toward a particular specialized cell type is demanding and may increase risks of neoplasia and undesired cell types. Precursor/progenitor cells from the organ of therapeutic concern typically lack only one critical attribute--the capacity for sustained self-renewal. We speculated that this could be induced in a regulatable manner such that cells proliferate only in vitro and differentiate in vivo without the need for promoting pluripotence or specifying lineage identity. As proof-of-concept, we generated and tested the efficiency, safety, engraftability, and therapeutic utility of "induced conditional self-renewing progenitor (ICSP) cells" derived from the human central nervous system (CNS); we conditionally induced self-renewal efficiently within neural progenitors solely by introducing v-myc tightly regulated by a tetracycline (Tet)-on gene expression system. Tet in the culture medium activated myc transcription and translation, allowing efficient expansion of homogeneous, clonal, karyotypically normal human CNS precursors ex vivo; in vivo, where Tet was absent, myc was not expressed, and self-renewal was entirely inactivated (as was tumorigenic potential). Cell proliferation ceased, and differentiation into electrophysiologically active neurons and other CNS cell types in vivo ensued upon transplantation into rats, both during development and after adult injury--with functional improvement and without neoplasia, overgrowth, deformation, emergence of non-neural cell types, phenotypic or genomic instability, or need for immunosuppression. This strategy of inducing self-renewal might be applied to progenitors from other organs and may prove to be a safe, effective, efficient, and practical method for optimizing insights gained from the ability to reprogram cells.

Dorsomorphin Promotes Human Embryonic Stem Cell Self-renewal

Angewandte Chemie (International Ed. in English). Apr, 2011  |  Pubmed ID: 21384475

Long-term Magnetic Resonance Imaging of Stem Cells in Neonatal Ischemic Injury

Annals of Neurology. Feb, 2011  |  Pubmed ID: 21387373

Quantitative magnetic resonance imaging (MRI) can serially and noninvasively assess the degree of injury in rat pup models of hypoxic ischemic injury (HII). It can also noninvasively monitor stem cell migration following iron oxide prelabeling. Reports have shown that neural stem cells (NSCs) may help mediate neuroprotection or stimulate neuroreparative responses in adult and neonatal models of ischemic injury. We investigated the ability of high-field MRI to monitor and noninvasively quantify the migration, proliferation, and location of iron oxide-labeled NSCs over very long time periods (58 weeks) in real time while contemporaneously correlating this activity with the evolving severity and extent of neural damage.

The Risk of Putting Something Where It Does Not Belong: Mesenchymal Stem Cells Produce Masses in the Brain

Experimental Neurology. Jul, 2011  |  Pubmed ID: 21420402

Overexpression of Basic Helix-Loop-Helix Transcription Factors Enhances Neuronal Differentiation of Fetal Human Neural Progenitor Cells in Various Ways

Stem Cells and Development. Jul, 2011  |  Pubmed ID: 21561385

In a perspective of regenerative medicine, multipotent human neural progenitor cells (hNPCs) offer a therapeutic advantage over pluripotent stem cells in that they are already invariantly "neurally committed" and lack tumorigenicity. However, some of their intrinsic properties, such as slow differentiation and uncontrolled multipotency, remain among the obstacles to their routine use for transplantation. Although rodent NPCs have been genetically modified in vitro to overcome some of these limitations, the translation of this strategy to human cells remains in its early stages. In the present study, we compare the actions of 4 basic helix-loop-helix transcription factors on the proliferation, specification, and terminal differentiation of hNPCs isolated from the fetal dorsal telencephalon. Consistent with their proneural activity, Ngn1, Ngn2, Ngn3, and Mash1 prompted rapid commitment of the cells. The Ngns induced a decrease in proliferation, whereas Mash1 maintained committed progenitors in a proliferative state. As opposed to Ngn1 and Ngn3, which had no effect on glial differentiation, Ngn2 induced an increase in astrocytes in addition to neurons, whereas Mash1 led to both neuronal and oligodendroglial specification. GABAergic, cholinergic, and motor neuron differentiations were considerably increased by overexpression of Ngn2 and, to a lesser extent, of Ngn3 and Mash1. Thus, we provide evidence that hNPCs can be efficiently, rapidly, and safely expanded in vitro as well as rapidly differentiated toward mature neural (typically neuronal) lineages by the overexpression of select proneural genes.

The Intricate Dance Between Repair and Inflammation: Introduction to Special Issue

Experimental Neurology. Jul, 2011  |  Pubmed ID: 21596040

DMSO-Free Programmed Cryopreservation of Fully Dissociated and Adherent Human Induced Pluripotent Stem Cells

Stem Cells International. 2011  |  Pubmed ID: 21716669

THREE MODES FOR CRYOPRESERVATION (CP) OF HUMAN IPSC CELLS HAVE BEEN COMPARED: STD: standard CP of small clumps with 10% of CPA in cryovials, ACC: dissociation of the cells with Accutase and freezing in cryovials, and PLT: programmed freezing of adherent cells in plastic multiwell dishes in a programmable freezer using one- and multistep cooling protocols. Four CPAs were tesetd: dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PG), and glycerol (GLY). The cells in ACC and PLT were frozen and recovered after thawing in the presence of a ROCK inhibitor Y-27632 (RI). EG was less toxic w/o CP cryopreservation than DMSO and allowed much better maintenance of pluripotency after CP than PG or GLY. The cells were cryopreserved very efficiently as adherent cultures (+RI) in plates (5-6-fold higher than STD) using EG and a 6-step freezing protocol. Recovery under these conditions is comparable or even higher than ACC+RI. Conclusions. Maintenance of cell-substratum adherence is a favorable environment that mitigates freezing and thawing stresses (ComfortFreeze(®) concept developed by CELLTRONIX). CP of cells directly in plates in ready-to-go after thawing format for HT/HC screening can be beneficial in many SC-related scientific and commercial applications such as drug discovery and toxicity tests.

Modeling Complex Neuropsychiatric Disorders with Human Induced Pluripotent Stem Cells

Current Opinion in Pharmacology. Oct, 2011  |  Pubmed ID: 21723195

Identifying the molecular and cellular basis of complex neuropsychiatric disorders (cNPDs) has been limited by the inaccessibility of central neurons, variability within broad diagnostic classifications, and the interplay of genetic and environmental factors. Recent work utilizing neuronally differentiated human induced pluripotent stem cells (hiPSCs) from Mendelian and polygenic cNPDs is beginning to illuminate neuritic, synaptic or cell body variations accompanied by specific gene or protein expression alterations largely mimicking known pathology. In some cases, phenotypes have only emerged after application of cellular stress or long duration of differentiation. Pathological and cellular expression features are fully or partially responsive to pharmacological treatment highlighting the potential utility of differentiated hiPSCs for discovery of personalized therapeutics and for identifying pathogenetically relevant targets in subgroups of patients within a broad syndromic classification. Because of the inherent variability in developing and differentiating hiPSC lines and the multiple comparisons implicit in 'omics' technologies, rigorous algorithms for assuring statistical significance and independent confirmation of results, will be required for robust modeling of cNPDs.

Phosphoproteomic Analysis: An Emerging Role in Deciphering Cellular Signaling in Human Embryonic Stem Cells and Their Differentiated Derivatives

Stem Cell Reviews. Oct, 2011  |  Pubmed ID: 22009073

Cellular signaling is largely controlled by protein phosphorylation. This post-translational modification (PTM) has been extensively analyzed when examining one or a few protein phosphorylation events that effect cell signaling. However, protein kinase-driven signaling networks, comprising total (phospho)proteomes, largely control cell fate. Therefore, large-scale analysis of differentially regulated protein phosphorylation is central to elucidating complex cellular events, including maintenance of pluripotency and differentiation of embryonic stem cells (ESCs). The current technology of choice for total phosphoproteome and combined total proteome plus total phosphoproteome (termed (phospho)proteome) [1] analyses is multidimensional liquid chromatography- (MDLC) tandem mass spectrometry (MS/MS). Advances in the use of MDLC for separation of peptides comprising total (phospho)proteomes, phosphopeptide enrichment, separation of enriched fractions, and quantitative peptide identification by MS/MS have been rapid in recent years, as have improvements in the sensitivity, speed, and accuracy of mass spectrometers. Increasingly deep coverage of (phospho)proteomes is allowing an improved understanding of changes in protein phosphorylation networks as cells respond to stimuli and progress from one undifferentiated or differentiated state to another. Although MDLC-MS/MS studies are powerful, understanding the interpretation of the data is important, and targeted experimental pursuit of biological predictions provided by total (phospho)proteome analyses is needed. (Phospho)proteomic analyses of pluripotent stem cells are in their infancy at this time. However, such studies have already begun to contribute to an improved and accelerated understanding of basic pluripotent stem cell signaling and fate control, especially at the systems-biology level.