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Recent studies indicate that transplanted neural stem/progenitor cells (NSPs) can interact with the environment of the central nervous system and stimulate protection and regeneration of host cells exposed to oxidative stress. Here, a set of animals grafted with NSPs and treated with 3-nitropropionic acid (3-NP) exhibited reduced behavioral symptoms and less severe damage of striatal cytoarchitecture than sham transplanted controls including better survival of neurons. Sites of tissue sparing correlated with the distribution pattern of donor cells in the host brain. To investigate the cellular and molecular bases of this phenomenon, we treated cocultures of NSPs and primary neural cell cultures with 3-NP to induce oxidative stress and to study NSP-dependent activation of antioxidant mechanisms and cell survival. Proactive presence of NSPs significantly improved cell viability by interfering with production of free radicals and increasing the expression of neuroprotective factors. This process was accompanied by elevated expression of ciliary neurotrophic factor (CNTF) and vascular endothelial growth factor (VEGF) in a network of NSPs and local astrocytes. Intriguingly, both in vitro and in vivo, enhanced growth factor secretion stimulated a robust upregulation of the antioxidant enzyme superoxide dismutase 2 (SOD2) in neurons and resulted in their improved survival. Our findings thus reveal a so far unrecognized mechanism of interaction between NSPs and surrounding cells accompanying neuroprotection: through mutual, NSP-triggered stimulation of growth factor production and activation of antioxidant mechanisms, cellular networks may shield the local environment from the arriving impact of oxidative stress. Disclosure of potential conflicts of interest is found at the end of this article.

Although the potential value of transplanted and endogenous neural stem cells (NSCs) for the treatment of the impaired central nervous system (CNS) has widely been accepted, almost nothing is known about their sensitivity to the hostile microenvironment in comparison to surrounding, more mature cell populations. Since many neuropathological insults are accompanied by oxidative stress, this report compared the alertness of antioxidant defense mechanisms and cell survival in NSCs and postmitotic neural cells (PNCs). Both primary and immortalized cells were analyzed. At steady state, NSCs distinguished themselves in their basal mitochondrial metabolism from PNCs by their lower reactive oxygen species (ROS) levels and higher expression of the key antioxidant enzymes uncoupling protein 2 (UCP2) and glutathione peroxidase (GPx). Following exposure to the mitochondrial toxin 3-nitropropionic acid, PNC cultures were marked by rapidly decreasing mitochondrial activity and increasing ROS content, both entailing complete cell loss. NSCs, in contrast, reacted by fast upregulation of UCP2, GPx, and superoxide dismutase 2 and successfully recovered from an initial deterioration. This recovery could be abolished by specific antioxidant inhibition. Similar differences between NSCs and PNCs regarding redox control efficiency were detected in both primary and immortalized cells. Our first in vivo data from the subventricular stem cell niche of the adult mouse forebrain corroborated the above observations and revealed strong baseline expression of UCP2 and GPx in the resident, proliferating NSCs. Thus, an increased "vigilance" of antioxidant mechanisms might represent an innate characteristic of NSCs, which not only defines their cell fate, but also helps them to encounter oxidative stress in diseased CNS.

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.

Here, we present our preliminary data showing that neural stem cells (NSCs) can prevent the degeneration of striatal neurons when transplanted into the CNS prior to intoxication with 3-nitropropionic acid (3-NP). In the adult CNS, the number of NSCs, a major source of neural cell populations and plasticity-modulating factors, is relatively low if compared to that of the developing brain. This, together with the adult growth-inhibitory environment, limits its regenerative capacity. Our recent observation has shown that grafted NSCs may rescue/protect neurons in the chronically impaired mesostriatal system. On the basis of this study and because we were also intrigued by our recent observations regarding the rescue/protective role of NSCs in vitro, we decided to test the hypothesis that grafted NSCs can also be deposited preventively in the CNS (and perhaps join the pool of endogenous NSCs of the intact host brain) for later buffering and maintenance of homeostasis when the host is exposed to oxidative stress.

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.

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.

In this review, we explore the possibility that, in many neurotransplantation paradigms, fetal organotypic explants and neural stem cells may – in addition to cell replacement and gene therapy – exert a therapeutic influence upon damaged host central nervous system tissue by an under-appreciated third mechanism – their inherent capacity to promote a regenerative response in the host. We postulate that this action is actually a fortunate byproduct of fundamental aspects of stem cell behavior in developing neural tissue.

Cell-based therapies such as neural transplantation have, until recently, been reserved for focal or regionally restricted neurologic diseases. These are best exemplified by Parkinson’s disease, in which encouraging progress in the use of neural transplantation, especially the grafting of fetal tissue, has been made experimentally (1,2) and clinically (3). [Even recent clinical studies that seemed to call into question such efficacy indicated that implanted fetal cells do exert a local impact albeit one that seemed to provoke an “overdose” effect (4)]. Donor tissue replaces dopamine via the engraftment and enhanced survival of neurotransmitter-secreting cells within the striatum or by forestalling degeneration of dopaminergic cells within the substantia nigra. However, the pathologic lesions of most neurogenetic diseases—indeed, most neurologic disorders—are usually widely disseminated in the brain and spinal cord and have not typically been regarded as within the purview of neural transplantation. Such diseases include not only the inherited neurodegenerative diseases of the pediatric age group (e.g., the lysosomal storage diseases, the leukodystrophies, inborn errors of metabolism, hypoxic—ischemic encephalopathy) but also such adult maladies as Alzheimer’s disease (AD), Huntington’s disease (HD), multi-infarct dementia, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), and brain tumors (especially glioblastomas). Therapeutic approaches for such “global” problems have typically depended on pharmacologic or genetic interventions; they have been regarded as beyond the purview of cellular-mediated approaches. Cell replacement therapies have largely been limited to transplantation of somatic cells derived from the hematopoietic system administered via bone marrow transplantation (BMT). In the majority of these disorders, such strategies have been unsatisfactory for treating the central nervous system (CNS) component of the disease.

This chapter reviews some of the work that has been performed in animal models of CNS diseases, where transplanted neural stem cells (NSCs) have mediated a therapeutic effect. Despite the presence of endogenous NSCs in the mammalian brain, it is recognized that intrinsic “self-repair” activity for the most devastating of injuries is inadequate or ineffective. This poor “regenerative” ability, particularly in the adult CNS, may be because of the limited number and restricted location of native NSCs and/or limitations imposed by the surrounding microenvironment, which may not be supportive or instructive for neuronal differentiation. Several transplantation experiments have suggested that neurogenic cues are transiently elaborated during degenerative processes and that exogenous NSCs are able to sense, home in, and respond appropriately. Neural stem cells display extensive tropism for pathology in an adult brain and can express bioactive genes within such pathological situations: evidence from intracranial gliomas. NSCs migrate extensively throughout a brain tumor mass in vivo and “trail” advancing tumor cells. A major requirement for the better use of NSCs is a better understanding of the pathophysiology of the diseases to be targeted—that is, knowing what aspects require repair and which cell type or types require replacement or rescue. A better understanding of fundamental NSC biology is required before human NSCs can be transplanted efficaciously in true clinical settings.

The cell recognition molecule L1, of the immunoglobulin superfamily, participates in the formation of the nervous system and has been shown to enhance cell migration and neurite outgrowth in vitro. To test whether ectopic expression of L1 would influence axonal outgrowth in vivo, we studied the development of the corticospinal tract in transgenic mice expressing L1 in astrocytes under the control of the GFAP-promoter. Corticospinal axons innervate their targets by extending collateral branches interstitially along the axon shaft following a precise spatio-temporal pattern. Using DiI as an anterograde tracer, we found that in the transgenic animals, corticospinal axons appear to be defasciculated, reach their targets sooner and form collateral branches innervating the basilar pons at earlier developmental stages and more diffusely than in wild type littermates. Collateral branches in the transgenic mice did not start out as distinct rostral and caudal sets, but they branched from the axon segments in a continuous rostrocaudal direction across the entire region of the corticospinal tract overlying the basilar pons. The ectopic branches are transient and no longer present at postnatal day 22. The earlier outgrowth and altered branching pattern of corticospinal axons in the transgenics is accompanied by an earlier differentiation of astrocytes. Taken together, our observations provide evidence that the ectopic expression of L1 on astrocytes causes an earlier differentiation of these cells, results in faster progression of corticospinal axons and influences the branching pattern of corticospinal axons innervating the basilar pons.

In recent years, it has become evident that the developing and even the adult mammalian central nervous system contains a population of undifferentiated, multipotent cell precursors, neural stem cells, the plastic properties of which might be of advantage for the design of more effective therapies for many neurological diseases. This article reviews the recent progress in establishing rodent and human clonal neural stem cell lines, their biological properties, and how these cells can be utilized to a correct variety of defects, with prospects for the near future to harness their behaviour for neural stem cell-based treatment of diseases in humans.

Remodeling of the mechanically injured cerebral cortex of kittens was studied in the presence of a neural xenograft taken from mouse fetuses. Solid neural tissue from the neopallium of a 14-day-old fetus was transferred into a cavity prepared in visual cortical area 18 of 33-day-old kittens. Injections of bromodeoxyuridine (BrdU) were used to monitor postoperative cell proliferation. Two months after transplantation, the presence of graft tissue in the recipient brain was assessed by Thy-1 immunohistochemistry. Antibodies specific for neurons, astrocytes, and oligodendrocytes and hematoxylin staining for endothelial cells were used for the characterization of proliferating (BrdU+) cells. The following were the major observations: 1) Of ten transplanted kittens, four had the cavity completely filled with neural tissue that resembled the intact cerebral cortex in its cytoarchitecture, whereas, in four other kittens, the cavity was partially closed. In two kittens, the cavity remained or became larger, which was also the case with all four sham-operated (lesioned, without graft) animals. 2) A substantial part of the remodeled tissue was of host origin. Only a few donor cells survived and dispersed widely in the host parenchyme. 3) In the remodeled region of transplanted animals, the densities of nerve, glial, and endothelial cells were similar to those in intact animals. 4) Cell proliferation increased after transplantation but only within a limited time, because, 2 months after the operation, the number of mitotic cells in the grafted cerebral cortex did not differ from that in intact controls. Our data suggest that the xenograft evokes repair processes in the kitten visual cortex that lead to structural recovery from a mechanical insult. The regeneration seems to rely on a complex interplay of many different mechanisms, including attenuation of necrosis, cell proliferation, and immigration of host cells into the wounded area. J. Comp. Neurol. 395:91–111, 1998. © 1998 Wiley-Liss, Inc.

Repair of mechanically injured primary somatosensory cortex in 3 week old mice was studied by placing small, solid foetal neurotransplants into large cortical cavities. After transplantation, the graft and host tissues were distinguished immunocytochemically owing to their expression of two different Thy-1 antigens. Cell proliferation was monitored by 3H-thymidine autoradiography. The following observations were made two months after operation: (i) In 8 out of 11 grafted animals new cortical tissue had taken the place of the cavity. (ii) Five of these 8 animals contained only host tissue; the remainder presented a small piece of grafted tissue. (iii) In the restored cortical area, newly generated cells were predominantly of host origin. These data suggest that the restorative capacity of the already post-mitotic cerebral cortex is not lost and may be reactivated. The presence of a foetal neural graft seems to favour this process.

In recent years, it has become evident that the developing and even the adult mammalian CNS contain a population of undifferentiated, multipotent cell precursors, neural stem cells, the plastic properties of which might be of advantage for the design of more effective therapies for many neurological diseases. This article reviews the recent progress in establishing rodent and human clonal neural stem cell lines, their biological properties, and how these cells can be utilized to correct a variety of defects, with prospects for the near future to harness their behaviour for neural stem cell-based treatment of diseases in humans.

Pigs and miniature pigs are steadily gaining importance as large animal models in the field of regenerative medicine, including stem cell research. With their size, organ capacity, and physiology resembling in several aspects that of humans, pigs are well suited for preclinical experiments and long-term safety studies. In this chapter, we summarize our experience with the isolation and culture of several somatic stem cell populations from fetal and adult pig tissue and briefly review their potential usefulness in future stem cell-based therapies. We also provide protocols for the isolation of fetal porcine neural stem cells (NSCs), adult bone marrow mesenchymal stem cells (MSCs), and epidermal progenitor cells (EPCs) from adult hair follicles.

Stem cell biology, construed in its broadest sense, has forced Medicine to view development and disease, and subsequent potential therapies, from an entirely different perspective (1, 2, 3). We have learned that there is a n inborn lasticity anThe Burnham Institute,,La Jolla,CAd flexibility “programmed” into the organism and its organ systems (1). The repository of this plasticity is thought to be the stem cell—the most primordial cell in the body and in any given structure. Nearly two decades ago, investigators began to identify cells with surprising plasticity and a propensity for dynamically shifting their fates within cultures obtained from developing and mature organs (1). The existence of such cells challenged the prevailing dogma that organs were rigidly and immutably constructed. Stem cells, as these plastic cells came to be termed, began to garner the interest of the developmental community, as well as that of the repair, gene therapy, and transplant communities. This interest arose when it was recognized that stem cells could be expanded in number and reimplanted into organs, where they would reintegrate appropriately and seamlessly, shift their fate in response to local cues to compensate for the absence of cells, express new genes, and in some cases, help promote functional improvement in disease models (4, 5, 6, 7, 8, 9, 10, 11, 12, 13)( Fig. 1). Exploiting the power of a cell that presumably had a pivotal role in development for repair purposes is somewhat analogous to rebooting a computer or reseeding a lawn. Optimizing these natural processes is the primary focus of today’s regenerative medicine. There have been a wide range of compelling studies conducted in animal models using various stem cells, including models of aging, spinal cord injury, stroke, parkinsonism, amyotrophic lateral sclerosis (ALS), cancer, multiple sclerosis, blood diseases, immunodeficiencies, enzyme deficiencies, myocardial infarcts, and diabetes.

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 α-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.

A new light was shed on the utility of neural grafts when it was rec- ognized that donor tissues and cells offer more than a source of immature pro- genitors potentially capable of cell replacement: First, they have the inherent capacity to produce multiple trophic and tropic factors promoting cell survival and tissue plasticity often characteristic of the immature central nervous sys- tem (CNS). Second, by their interaction with the host microenvironment via cell/cell and cell/ECM interactions, these grafts are capable of re-establishing homeostasis, which can be, for example, reflected in rescue and protection of host elements from harmful influences. This second capacity of donor cells re- lies, in part, also on a "dormant" but still present regenerative capacity of ma- ture or even aged CNS and on the possibility of its mobilization in the damaged nervous system by neural grafts. For this to occur efficiently after transplanta- tion, a bi-directional dialogue between donor and host cells must gradually be established, in which both "partners" transmit signals (cell/cell contact, molec- ular messengers), "listen to" and "understand" each other and are able to react by modifying their own plasticity- and development-related programs. Thus, for the best possible recovery of functionality in the injured adult and aged nervous system, neurotransplantation must always try to find optimal condi- tions for all three of the mentioned qualities of neural grafts, especially for the protection and/or reactivation of neural circuitry embedded in non-neurogenic CNS areas. Once fully understood, this newly recognized aspect of neurotrans- plantation (and topic of this review) might, someday, even allow the recovery of systems that would otherwise be doomed, such as cognition- and experience- related circuitry.

Crucial prerequisites for the development of safe preclinical protocols in biomedical research are suitable animal models that would allow for human-related validation of valuable research information gathered from experimentation with lower mammals. In this sense, the miniature pig, sharing many physiological similarities with humans, offers several breeding and handling advantages (when compared to non-human primates), making it an optimal species for preclinical experimentation. The present review offers several examples taken from current research in the hope of convincing the reader that the porcine animal model has gained massively in importance in biomedical research during the last few years. The adduced examples are taken from the following fields of investigation: (a) the physiology of reproduction, where pig oocytes are being used to study chromosomal abnormalities (aneuploidy) in the adult human oocyte; (b) the generation of suitable organs for xenotransplantation using transgene expression in pig tissues; (c) the skin physiology and the treatment of skin defects using cell therapy-based approaches that take advantage of similarities between pig and human epidermis; and (d) neurotransplantation using porcine neural stem cells grafted into inbred miniature pigs as an alternative model to non-human primates xenografted with human cells.

In this review we trace back the history of an idea that takes a new approach in restorative neurotransplantation by focusing on the “multifaceted dialogue” between graft and host and assigns a central role to graft-evoked host plasticity. In several experimental examples ranging from the transfer of solid fetal tissue grafts into mechanical cortical injuries to deposits of neural stem cells into hemisectioned spinal cord, MPTP-damaged substantia nigra or mutant cerebella supportive evidence is provided for the hypothesis, that in many CNS disorders regeneration of the host CNS can be achieved by taking advantage of the inherent capacity of neural grafts to induce protective and restorative mechanisms within the host. This principle might once allow us to spare even complex circuitry from neurodegeneration.

Current restorative neurotransplantation research focuses mainly on the potential of the neural graft to replace damaged or missing cell populations and to deliver needed gene products in the form of transgenes. Because of this graft-oriented bias of the procedure, possible dormant regenerative capabilities within the host have been largely underestimated and dismissed as insignificant. This review discusses existing evidence that neural grafts can have stimulating effects on host-intrinsic plasticity that can help regeneration of the mammalian central nervous system. If confirmed, the synergistic interaction between graft and host might substantially enhance our therapeutic possibilities.

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.

Many central nervous system regions at all stages of life contain neural stem cells (NSCs). We explored how these disparate NSC pools might emerge. A traceable clone of human NSCs was implanted intraventricularly to allow its integration into cerebral germinal zones of Old World monkey fetuses. The NSCs distributed into two subpopulations: One contributed to corticogenesis by migrating along radial glia to temporally appropriate layers of the cortical plate and differentiating into lamina-appropriate neurons or glia; the other remained undifferentiated and contributed to a secondary germinal zone (the subventricular zone) with occasional members interspersed throughout brain parenchyma. An early neurogenetic program allocates the progeny of NSCs either immediately for organogenesis or to undifferentiated pools for later use in the “postdevelopmental” brain.