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To test the idea that genetically engineered cells can rescue axotomized neurons, we transplanted fibroblasts and immortalized neural stem cells (NSCs) modified to express neurotrophic factors into the injured spinal cord. The neurotrophin-3 (NT-3) or nerve growth factor (NGF) transgene was introduced into these cells using recombinant retroviral vectors containing an internal ribosome entry site (IRES) sequence and the b-galactosidase or alkaline phosphatase reporter gene. Bioassay confirmed biological activity of the secreted neurotrophic factors. Clarke’s nucleus (CN) axons, which project to the rostral spinal cord and cerebellum, were cut unilaterally in adult rats by T8 hemisection. Rats received transplants of fibroblasts or NSCs genetically modified to express NT-3 or NGF and a reporter gene, only a reporter gene, or no transplant. Two months postoperatively, grafted cells survived at the hemisection site. Grafted fibroblasts and NSCs expressed a reporter gene and immunoreactivity for the NGF or NT-3 transgene. Rats receiving no transplant or a transplant expressing only a reporter gene showed a 30% loss of CN neurons in the L1 segment on the lesioned side. NGF-expressing transplants produced partial rescue compared with hemisection alone. There was no significant neuron loss in rats receiving grafts of either fibroblasts or NSCs engineered to express NT-3. We postulate that NT-3 mediates survival of CN neurons through interaction with trkC receptors, which are expressed on CN neurons. These results support the idea that NT-3 contributes to long-term survival of axotomized CN neurons and show that genetically modified cells rescue axotomized neurons as efficiently as fetal CNS transplants. J. Neurosci. Res. 65:549 ‐564, 2001. © 2001 Wiley-Liss, Inc.

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.

Human-induced pluripotent stem cells (hiPSCs) derived from somatic cells of patients have opened possibilities for in vitro modeling of the physiology of neural (and other) cells in psychiatric disease states. Issues in early stages of technology development include (1) establishing a library of cells from adequately phenotyped patients, (2) streamlining laborious, costly hiPSC derivation and characterization, (3) assessing whether mutations or other alterations introduced by reprogramming confound interpretation, (4) developing efficient differentiation strategies to relevant cell types, (5) identifying discernible cellular phenotypes meaningful for cyclic, stress induced or relapsing–remitting diseases, (6) converting phenotypes to screening assays suitable for genome-wide mechanistic studies or large collection compound testing and (7) controlling for variability in relation to disease specificity amidst low sample numbers. Coordination of material for reprogramming from patients well-characterized clinically, genetically and with neuroimaging are beginning, and initial studies have begun to identify cellular phenotypes. Finally, several psychiatric drugs have been found to alter reprogramming efficiency in vitro, suggesting further complexity in applying hiPSCs to psychiatric diseases or that some drugs influence neural differentiation moreso than generally recognized. Despite these challenges, studies utilizing hiPSCs may eventually serve to fill essential niches in the translational pipeline for the discovery of new therapeutics.

Stem cell-based therapies are viewed as among the most promising new strategies against developmental, traumatic, oncogenic and degenerative diseases. Indeed, it was the ability to identify, isolate, culture, interrogate, expand and transplant stem cells and/or their derivatives that gave rise to the new field of ‘regenerative medicine'. While the hope for stem cell-based regenerative medicine grows rapidly, it is becoming clear from extant clinical trials that we still need a greater understanding of the fundamentals of stem cell biology as well as of the disease process in order to make an impact that is efficient, efficacious, safe, reliable, scalable, cost-effective, practical, accessible and affordable. During the past 20 years, the breadth of what is regarded as ‘stem cell research' has expanded immensely, now embracing, for example, tissue-specific, embryonic and induced/reprogrammed stem cells as well as cancer stem cells. Moreover, in the past 3–5 years, clinical trials using or relying on exogenous or endogenous stem cells (including cancer stem cells) have been launched with many more on the drawing board. With such rapid advances in the field, we felt it necessary to concisely take an overview of current thinking in stem cell biology, with a particular eye to potential applications. In this special issue of Experimental & Molecular Medicine, we offer concise coverage of the state-of-the art in a series of representative areas in stem cell research provided by thought leaders in each topic. First, the developmental changes in the characteristics of hematopoietic stem cells (HSCs), the one cobble stone model of tissue-specific stem cells, is discussed by Dr C Eaves. HSCs undergo extensive changes during development in their proliferation, self-renewal and growth factor responsiveness as well as their regenerative capacity. While the mechanisms underlying the shift in HSC properties remain largely unclear, this paper provides a recent view on the miRNA-mediated regulatory mechanisms that can define the developmental changes in HSCs. Another type of stem cell, one that is sometimes shrouded in controversy, ‘very small embryonic-like stem cells (VSELs)', is discussed by Dr M Ratajczak. Dr Ratajczak addresses this controversy by describing their isolation, in-vivo identity and evidence for pluripotency based on transcriptomics and multi-lineage differentiation potential. Dr E Snyder's group provides a recent view on the use of human-induced pluripotent stem cells (hiPSCs) for understanding the mechanisms underlying a complex disease (for example, bipolar disorder) based on responsiveness to a known clinically efficacious drug (for example, lithium). Based on a better understanding of lithium's heretofore unknown mechanism-of-action, then the same hiPSCs may be used to help screen and identify drugs that work better and with less toxicity than lithium against these same targets. In other words, in addition to being potentially useful for autologous cell therapy, hiPSCs can also serve as powerful tools for drug discovery based on their ability to be obtained from patients that harbor a disease and yield cells in a particular lineage that presumably also ‘have that disease'. Moving to more applied stem cell biology, Dr A Caplan provides a concise review of mesenchymal stromal cell (MSC)-based therapy, describing basic concept as well as a mode of therapeutic action based on the secretion of factors that facilitate establishment of a regenerative microenvironment. Dr Caplan offers a spectrum of clinical studies ranging from immunological disorders to organ regeneration that have employed MSCs. Dr J Yoo provides insight on the use of tissue-engineering scaffolds to facilitate a regenerative microenvironment in injured organs by stimulating the recruitment and proliferation of endogenous stem cells. In addition, the current edition provides representative original researches covering each area of stem cell biology and therapeutic applications. The feasibility of using stem cell therapy in the treatment of neuronal diseases using neuronal stem cells and embryonic stem cell-derived neuronal progenitor cells as model systems is explored. Strategies for recruiting endogenous stem cells during wound healing and the possibility of salivary gland regeneration was explored. Finally, the clinical efficacy of stem cell therapy for femoral head necrosis was evaluated for 128 patients after 5 years of follow-up is discussed. Collectively, these articles not only provide an overview of the state-of-the-art and future prospects for stem cell biology, but also describe the pipeline required to enable stem cell research to impact regenerative medicine. We are particularly thankful to Dr Dae-Myung Jue, the Editor-in-Chief of Experimental & Molecular Medicine and to the Nature Publishing Group for their support on this special series ‘Special Feature on Stem Cells: Current Research and Future Prospects'.

One of the impediments to the treatment of brain tumors (e.g., gliomas) has been the degree to which they expand, infiltrate surrounding tissue, and migrate widely into normal brain, usually rendering them “elusive” to effective resection, irradiation, chemotherapy, or gene therapy. We demonstrate that neural stem cells (NSCs), when implanted into experimental intracranial gliomas in vivo in adult rodents, distribute themselves quickly and extensively throughout the tumor bed and migrate uniquely in juxtaposition to widely expanding and aggressively advancing tumor cells, while continuing to stably express a foreign gene. The NSCs “surround” the invading tumor border while “chasing down” infiltrating tumor cells. When implanted intracranially at distant sites from the tumor (e.g., into normal tissue, into the contralateral hemisphere, or into the cerebral ventricles), the donor cells migrate through normal tissue targeting the tumor cells (including human glioblastomas). When implanted outside the CNS intravascularly, NSCs will target an intracranial tumor. NSCs can deliver a therapeutically relevant molecule—cytosine deaminase—such that quantifiable reduction in tumor burden results. These data suggest the adjunctive use of inherently migratory NSCs as a delivery vehicle for targeting therapeutic genes and vectors to refractory, migratory, invasive brain tumors. More broadly, they suggest that NSC migration can be extensive, even in the adult brain and along nonstereotypical routes, if pathology (as modeled here by tumor) is present.

Cell transplantation into host brain requires a reliable cell marker to trace lineage and location of grafted cells in host tissue. The lacZ gene encodes the bacterial (E. coli) enzyme β-galactosidase (β-gal) and is commonly visualized as a blue intracellular precipitate following its incubation with a substrate, “X-gal,” in an oxidation reaction. LacZ is the “reporter gene” most commonly employed to follow gene expression in neural tissue or to track the fate of transplanted exogenous cells. If the reaction is not performed carefully—with adequate optimization and individualization of various parameters (e.g., pH, concentration of reagents, addition of chelators, composition of fixatives) and the establishment of various controls—then misleading nonspecific background X-gal positivity can result, leading to the misidentification of cells. Some of this background results from endogenous nonbacterial β-gal activity in discrete populations of neurons in the mammalian brain; some results from an excessive oxidation reaction. Surprisingly, few articles have emphasized how to recognize and to eliminate these potential confounding artifacts in order to maximize the utility and credibility of this histochemical technique as a cell marker. We briefly review the phenomenon in general, discuss a specific case that illustrates how an insufficiently scrutinized X-gal positivity can be a pitfall in cell transplantation studies, and then provide recommendations for optimizing the specificity and reliability of this histochemical reaction for discerning E. coli β-gal activity.

The ready availability of unlimited quantities of neural stem cells derived from the human brain holds great interest for basic and applied neuroscience, including therapeutic cell replacement and gene transfer following transplantation. We report here the combination of epigenetic and genetic procedures for perpetuating human neural stem cell lines. Thus we tested various culture conditions and genes for those that optimally allow for the continuous, rapid expansion and passaging of human neural stem cells. Among them, v-myc (the p110 gag–myc fusion protein derived from the avian retroviral genome) seems to be the most effective gene; we have also identified a strict requirement for the presence of mitogens (FGF-2 and EGF) in the growth medium, in effect constituting a conditional perpetuality or immortalization. A monoclonal, nestin-positive, human neural stem cell line (HNSC.100) perpetuated in this way divides every 40 h and stops dividing upon mitogen removal, undergoing spontaneous morphological differentiation and upregulating markers of the three fundamental lineages in the CNS (neurons, astrocytes, and oligodendrocytes). HNSC.100 cells therefore retain basic features of epigenetically expanded human neural stem cells. Clonal analysis confirmed the stability, multipotency, and self-renewability of the cell line. Finally, HNSC.100 can be transfected and transduced using a variety of procedures and genes encoding proteins for marking purposes and of therapeutic interest (e.g., human tyrosine hydroxylase I).

A dual scaffold structure made of biodegradable polymers and seeded with neural stem cells has been developed to address the issues of spinal cord injury including axonal severance and the loss of neurons and glia. The general design of the scaffold is derived the structure of the spinal cord with an outer section which mimics the white matter with long axial pores to provide axonal guidance and an inner section seeded with neural stem cells to address the issues of cell replacement and mimic the general character of the gray matter. The seeded scaffold leads to improved functional recovery as compared with the lesion control or cells alone following spinal cord injury.

Neural stem cells have been shown to participate in the repair of experimental CNS disorders. To examine their potential in spinal cord repair, we used retroviral vectors to genetically modify a clone of neural stem cells, C17, to overproduce neurotrophin-3 (NT-3). The cells were infected with a retrovirus construct containing the NT-3.IRES.lacZ/neo sequence and cloned by limiting dilution and selection for lacZ expression. We studied the characteristics of the modified neural stem cells in vitro and after transplantation into the intact spinal cord of immunosuppressed adult rats. Our results show that: (i) most of the genetically modified cells express both NT-3 and lacZ genes with a high coexpression ratio in vitro and after transplantation; and (ii) large numbers of the xenografted cells survive in the spinal cord of adult rats for at least 2 months, differentiate into neuronal and glial phenotypes, and migrate for long distances. We conclude that genetically modified neural stem cells, acting as a source of neurotrophic factors, have the potential to participate in spinal cord repair.

The implementation of neural stem cell lines as a source material for brain tissue transplants is currently limited by the ability to induce specific neurochemical phenotypes in these cells. Here, we show that coordinated induction of a ventral mesencephalic dopaminergic phenotype in an immortalized multipotent neural stem cell line can be achieved in vitro. This process requires both the overexpression of the nuclear receptor Nurr1 and factors derived from local type 1 astrocytes. Over 80% of cells obtained by this method demonstrate a phenotype indistinguishable from that of endogenous dopaminergic neurons. Moreover, this procedure yields an unlimited number of cells that can engraft in vivo and that may constitute a useful source material for neuronal replacement in Parkinson's disease.

The induction of spongiform myeloencephalopathy by murine leukemia viruses is mediated primarily by infection of central nervous system (CNS) microglia. In this regard, we have previously shown that CasBrE-induced disease requires late, rather than early, virus replication events in microglial cells (W. P. Lynch et al., J. Virol. 70:8896–8907, 1996). Furthermore, neurodegeneration requires the presence of unique sequences within the viral env gene. Thus, the neurodegeneration-inducing events could result from microglial expression of retroviral envelope protein alone or from the interaction of envelope protein with other viral structural proteins in the virus assembly and maturation process. To distinguish between these possible mechanisms of disease induction, we engineered the engraftable neural stem cell line C17-2 into packaging/producer cells in order to deliver the neurovirulent CasBrE env gene to endogenous CNS cells. This strategy resulted in significant CasBrE env expression within CNS microglia without the appearance of replication competent virus. CasBrE envelope expression within microglia was accompanied by increased expression of activation markers F4/80 and Mac-1 (CD11b) but failed to induce spongiform neurodegenerative changes. These results suggest that envelope expression alone within microglia is not sufficient to induce neurodegeneration. Rather, microglia-mediated disease appears to require neurovirulent Env protein interaction with other viral proteins during assembly or maturation. More broadly, the results presented here prove the efficacy of a novel method by which neural stem cell biology may be harnessed for genetically manipulating the CNS, not only for studying neurodegeneration but also as a paradigm for the disseminated distribution of retroviral vector-transduced genes.

Many diseases of the central nervous system (CNS), particularly those of genetic, metabolic, or infectious/inflammatory etiology, are characterized by “global” neural degeneration or dysfunction. Therapy might require widespread neural cell replacement, a challenge not regarded conventionally as amenable to neural transplantation. Mouse mutants characterized by CNS-wide white matter disease provide ideal models for testing the hypothesis that neural stem cell transplantation might compensate for defective neural cell types in neuropathologies requiring cell replacement throughout the brain. The oligodendrocytes of the dysmyelinated shiverer ( shi ) mouse are “globally” dysfunctional because they lack myelin basic protein (MBP) essential for effective myelination. Therapy, therefore, requires widespread replacement with MBP-expressing oligodendrocytes. Clonal neural stem cells transplanted at birth—using a simple intracerebroventricular implantation technique—resulted in widespread engraftment throughout the shi brain with repletion of MBP. Accordingly, of the many donor cells that differentiated into oligodendroglia—there appeared to be a shift in the fate of these multipotent cells toward an oligodendroglial fate—a subgroup myelinated up to 52% (mean = ≈40%) of host neuronal processes with better compacted myelin of a thickness and periodicity more closely approximating normal. A number of recipient animals evinced decrement in their symptomatic tremor. Therefore, “global” neural cell replacement seems feasible for some CNS pathologies if cells with stem-like features are used.

Publisher Summary A subpopulation of progenitors or stem cells exists in the mammalian central nervous system (CNS) whose isolation, propagation, and transplantation have clinical utility for molecular support therapy and/or cell replacement for some degenerative, developmental, and acquired insults to the CNS. Most neurologic diseases are characterized by complex pathology, often requiring multiple repair strategies. By virtue of their inherent biologic properties, CNS stem cells seem to possess multiple therapeutic capabilities. A number of options are possible from the implantation of neural stem cells into the dysfunctional CNS. First, transplanted stem cells may differentiate into and replace damaged or degenerated neurons and/or glia. Host cells degenerating not only by cell-intrinsic disease processes but also cell-extrinsic forces might theoretically be replaced. Because stem cells may incorporate into host cytoarchitecture in a functional manner, they may prove more than vehicles for “passive” delivery of substances: The regulated release of certain substances through feedback loops may be reconstituted, as might the reformation of essential connections. Although presently available gene transfer vectors usually depend on relaying new genetic information through established neural circuits, which may, in fact, have degenerated, neural stem cells may actually also reconstitute the pathways.

Stable clones of neural stem cells (NSCs) have been isolated from the human fetal telencephalon. These self-renewing clones give rise to all fundamental neural lineages in vitro. Following transplantation into germinal zones of the newborn mouse brain they participate in aspects of normal development, including migration along established migratory pathways to disseminated central nervous system regions, differentiation into multiple developmentally and regionally appropriate cell types, and nondisruptive interspersion with host progenitors and their progeny. These human NSCs can be genetically engineered and are capable of expressing foreign transgenes in vivo. Supporting their gene therapy potential, secretory products from NSCs can correct a prototypical genetic metabolic defect in neurons and glia in vitro. The human NSCs can also replace specific deficient neuronal populations. Cryopreservable human NSCs may be propagated by both epigenetic and genetic means that are comparably safe and effective. By analogy to rodent NSCs, these observations may allow the development of NSC transplantation for a range of disorders.

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.

GREEN fluorescent protein (GFP) is an effective marker for retrovirus and herpes virus vector-mediated gene transfer into various central nervous system-derived cells, both proliferative and non-proliferative, in culture and in vivo. Retrovirus vectors were used to stably transduce several rat and human glioma lines, and a multi-potent mouse neural progenitor line in culture. Implantation of selected pools of transduced glioma cells into rodent brain allowed clear visualization of the tumor and the invading tumor edge. Helper virus-free HSV-1 amplicon vectors successfully transferred gfp into non-dividing primary neural cells in culture and in the rat brain. This study describes the versatility of GFP for: (i) labelling of glioma cells in experimental brain tumor models and neural progenitor cells by retrovirus vectors, and (ii) efficient, non-toxic delivery of genes to post mitotic cells of the nervous system using helper-virus free HSV-1 amplicon vectors.

When neural progenitors are immortalized, they appear in many ways to behave as neural stem cells. In fact, they have been called “stem-like cells.” We have demonstrated that immortalized, multipotent, clonal neural progenitors or stem-like cells can, following transplantation, integrate appropriately into recipient mouse nervous system (from embryo to adult) and differentiate into a range of neurons and glia throughout the neuraxis. Donor progenitors appear to intermingle nondisruptively with endogenous progenitors and respond to many of the same spatial and temporal developmental signals in the same manner as host progenitors. Immortalization does not abrogate their ability to respond to normal cues (e.g. withdraw from the cell cycle, differentiate, intract with host cells). Because engrafted progenitors are recognized by expression of retrovirally/transduced lacZ, these data also support the feasibility of transplanting neural stem-like cells — expressing genes of developmental or therapeutic importance (either intrinsically or following ex vivo genetic manipulation) — as a strategy for sustained delivery of such factors directly to the CNS as integral members of the cytoarchitecture. Furthermore, progenitors might also replace injured cells and/or provide nondiffusible factors (myelin, “bridges,” cell-cell contact signals, extracellular matrix) that might allow the injured host to reform its own connections. Some of our studies have suggested successful therapeutic gene expression throughout the brains of mouse models of genetic metabolic neurodegenerative disease; repletion of degenerated or developmentally impaired neurons in new-born and adult mouse cerebrum and spinal cord; and differentiation into oligodendroglia that improve myelination in mouse mutants with diffuse white matter disease. We suggest that the inherent biologic properties of neural progenitors make them appealingly suited to be efficient, multifaceted vehicles for cell replacement, repair, and gene transfer in CNS dysfuncion. Their apparent ability to address widespread neuropathology is intriguing because most neurologic disease is not focal, but rather multifocal or global. The use of neural stem-like cells may, therefore, allow a cellular therapy like transplantation to address those therapeutic challenges usually consigned to pharmacologic or genetic interventions. Such stem-like cell lines may also provide models for the study of the commitment, differentation, and plasticity of neural progenitors and stem cells. We have hypothesized that some multipotent neural stem cells may have intrinsic ferentiation programs that may nevertheless be influenced by appropriately timed microenvironmental factors. Therefore, a system such as this may facilitate exploration of the reciprocal interactions between external cues and instrinsic programs.

Chondrocytes have been generated in vitro from a range of progenitor cell types and by a number of strategies. However, achieving reconstitution of actual physiologically relevant, appropriately-laminated cartilage in situ that would be applicable to conditions, such as arthritis and cartilage degeneration remains elusive. This lack of success is multifactorial and includes limited cell source, decreased proliferation rate of mature chondrocytes, lack of maintenance of phenotype, reduced matrix synthesis, and poor integration with host tissue. We report an efficient approach for deriving mesenchymal chondroprogenitor cells from human embryonic stem cells. These cells generated tissue containing cartilage-specific matrix proteins that integrated in situ in a partial-thickness defect in ex vivo articular cartilage harvested from human arthritic joints. Given that stem cells provide a virtually inexhaustible supply of starting material and that our technique is easily scalable, cartilaginous tissue primed and grafted in this manner could be suitable for clinical translation.

Many methods of gene transfer to the brain are under study for the treatment of the central nervous system manifestations of a number of diseases. One strategy employs vectors--typically genetically altered viruses--for the delivery of exogenous genes directly to a host's brain cells in situ. Alternatively, transplanting cells--of either neural or nonneural origin--that intrinsically secrete missing or therapeutic gene produces, or which are genetically engineered ex vivo to do so, may provide another strategy. Nonmigratory cells implanted into small, discrete anatomic sites are well suited for disease processes whose pathology is very focal. Neural cells that have the capacity to migrate in the central nervous system provide more powerful vehicles for delivering factors to disease involving pathology that is widely disseminated, extensive, multifocal, or even global (e.g., many neurogenetic diseases). Both neural and nonneural cell types have been successfully engineered to produce a variety of neurotransmitter synthetic enzymes, metabolic enzymes, and neurotrophic factors. Impressive results with genetically modified cells in animal models of central nervous system disease encourage the continued development of gene therapy strategies for treating neural dysfunction.

In humans, beta-hexosaminidase alpha-subunit deficiency prevents the formation of a functional beta-hexosaminidase A heterodimer resulting in the severe neurodegenerative disorder, Tay-Sachs disease. To explore the feasibility of using ex vivo gene transfer in this lysosomal storage disease, we produced ecotropic retroviruses encoding the human beta-hexosaminidase alpha-subunit cDNA and transduced multipotent neural cell lines. Transduced progenitors stably expressed and secreted high levels of biologically active beta-hexosaminidase A in vitro and cross-corrected the metabolic defect in a human Tay-Sachs fibroblasts cell line in vitro. These genetically engineered CNS progenitors were transplanted into the brains of both normal fetal and newborn mice. Engrafted brains, analyzed at various ages after transplant, produced substantial amounts of human beta-hexosaminidase alpha-subunit transcript and protein, which was enzymatically active throughout the brain at a level reported to be therapeutic in Tay-Sachs disease. These results have implications for treating neurologic diseases characterized by inherited single gene mutations.

Central nervous system (CNS)-derived neural cell lines have proven to be extremely useful for delineating mechanisms controlling such diverse phenomena as cell lineage choice and differentiation, synaptic maturation, neurotransmitter synthesis and release, and growth factor signalling. In addition, there has been hope that such lines might play pivotal roles in CNS gene therapy and repair. The ability of some neural cell lines to integrate normally into the CNS following transplantation and to express foreign, often corrective gene products in situ might offer potential therapeutic approaches to certain neurodegenerative diseases. Five general strategies have evolved to develop neural cell lines: isolation and cloning of spontaneous or mutagenically induced malignancies, targeted oncogenesis in transgenic mice, somatic cell fusion, growth factor mediated expansion of CNS progenitor or stem cells, and retroviral transduction of neuroepithelial precursors. in this article, we detail recent progress in these areas, focusing on those cell lines that have enabled novel insight into the mechanisms controlling neuronal cell lineage choice and differentiation, both in vitro and in vivo.

MANY metabolic diseases affecting the central nervous system are refractory to treatment because the blood–brain barrier restricts entry of therapeutic molecules. It may be possible to deliver therapeutic gene products directly to the brain by transplantation of neural progenitor cells, which can integrate into the murine central nervous system in a cytoarchitecturally appropriate manner1–7. We tested this approach in mucopolysaccharidosis VII (Sly disease), a lysosomal storage disorder of humans, dogs and mice caused by an inherited deficiency of β-glucuronidase8–10 . Lysosomal accumulation of glycosaminoglycans occurs in the brain and other tissues, causing a fatal progressive degenerative disorder, including mental retardation11. Treatments are designed to provide a source of normal enzyme for uptake by diseased cells12–20. We report here that by transplanting β-glucuronidase-expressing neural progenitors into the cerebral ventricles of newborn mice, donor cells engrafted throughout the neuraxis. At maturity, donor-derived cells were present as normal constituents of diverse brain regions. β-Glucu-ronidase activity was expressed along the entire neuraxis, resulting in widespread correction of lysosomal storage in neurons and glia in affected mice.

Publisher Summary This chapter focuses on retrovirally immortalized primary neural progenitors that give rise to neurons, particularly from the standpoint of transplantation into the mammalian central nervous system (CNS). Cell lines form an important part of the immortalization of neural cells. In a library of cell lines, Each line might represent cells plucked from defined developmental times and specific locations and could provide an unlimited source of homogeneous material from which factors could be purified and against which reagents could be assayed. The ability of immortalized neural progenitors to integrate normally into the nervous system following transplantation offers a potential therapeutic approach to certain neurodegenerative diseases. The genes that have been most successful at immortalization have been avian myc (v-myc) and SV40 large T antigen. These genes induce unlimited mitotic activity in vitro without promoting oncogenic qualities. There are two techniques that are used maintain progenitors, removed from the brain, in a proliferative state in vitro: transduction of immortalizing genes into neural progenitors and chronic exposure of progenitors to mitogenic cytokines. The sources of neural progenitors for transplant studies are neonatal cerebellum, embryonic and adult hippocampus, embryonic and adult striatum, and embryonic raphe nucleus. Technical aspects of grafting immortalized neural cells include labeling donor cells for engraftment and immune rejection, which can sometimes be rectified by cyclosporin immunosuppression of hosts. The chapter also discusses the strategies to test the ability of immortalized neural cell lines to effectively perform repairing by repopulating the cytoarchitecture of lesioned or mutant animals. Two other areas related to CNS dysfunction that have emerged as topics of interest are neural progenitor and stem cell biology with gene therapy and repair of the CNS via transplantation. Immortalized neural progenitors, when transplanted into the CNS, intermingle nondisruptively with endogenous progenitors and respond to the same spatial and temporal developmental signals similarly as host progenitors.

Individual neural progenitors, derived from the external germinal layer of neonatal murine cerebellum, were previously immortalized by the retrovirus-mediated transduction of avian myc (v-myc). C17-2 is one of those clonal multipotent progenitor cell lines (Snyder et al., 1992, Cell 68: 33-51; Ryder et al., 1990, J. Neurobiol. 21:356-375). When transplanted into newborn mouse cerebellum (CB), the cells participate in normal CB development; they engraft in a cytoarchitecturally appropriate, nontumorigenic manner and differentiate into multiple CB cell types (neuronal and glial) similar to endogenous progenitors (Snyder et al., 1992, as above). They also appear to engraft and participate in the development of multiple other structures along the neural axis and at multiple other stages (Snyder et al., 1993, Soc. Neurosci. Abstr. 19). Thus conclusions regarding these immortalized progenitors may be applicable to endogenous neural progenitors in vivo. To help identify and analyze factors that promote differentiation of endogenous progenitors, we first investigated the ability to maintain C17-2 cells in a defined, serum-free medium (N2). The cells survive in vitro in N2 but undergo mitosis at a very low rate. Addition of epidermal growth factor (EGF), however, either from mouse submaxillary gland or the human recombinant protein, appreciably stimulates thymidine incorporation and cell division approximately threefold. Basic fibroblast growth factor (bFGF) is an even more potent mitogen, promoting thymidine incorporation, cell division, and a net increase in cell number equal to that in serum. Both EGF and bFGF are active at very low nanomolar concentrations, suggesting that they interact with their respective receptors rather than a homologous receptor system. The findings demonstrate that C17-2 cells can be maintained and propagated in a fully defined medium, providing the basis for analysis of other growth and differentiation factors. That EGF and particularly bFGF are mitogenic for these cells is in accord with recent observations on primary neural tissue (Reynolds and Weiss, 1992, Science 255:1707-1710; Kilpatrick and Bartlett, 1993, Neuron 10:255-265; Ray et al., 1993, Proc. Natl. Acad. Sci. USA 90:3602-3606) suggesting that bFGF and EGF responsiveness may be fundamental properties of neural progenitors.

Multipotent neural cell lines were generated via retrovirus-mediated v-myc transfer into murine cerebellar progenitor cells. When transplanted back into the cerebellum of newborn mice, these cells integrated into the cerebellum in a nontumorigenic, cytoarchitecturally appropriate manner. Cells from the same clonal line differentiated into neurons or glia in a manner appropriate to their site of engraftment. Engrafted cells, identified by lacZ expression and PCR-mediated detection of a unique sequence arrangement, could be identified in animals up to 22 months postengraftment. Electron microscopic and immunohistochemical analysis demonstrated that some engrafted cells were similar to host neurons and glia. Some transplant-derived neurons received appropriate synapses and formed normal intercellular contacts. These data indicate that generating immortalized cell lines for repair of, or transport of genes into, the CNS may be feasible. Such lines may also provide a model for commitment and differentiation of cerebellar progenitor cells.

✓ 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.

Nat. Biotechnol. 24, 397–400 (2006); published online 29 March 2006; corrected after print 8 November 2006. In the version of this article initially published, on page 399, column 2, paragraph 4, the authors wrongly credited the idea for using cells from nonviable human embryos as an alternative source of human embryonic stem cells to Landry and Zucker (refs.7,26). The reference should have been to Alikani, M. & Willadsen, S.M., Reproductive BioMedicine Online 5, 56–58, 2002.

Nat Biotechnol. 22, 805–806 (2004) The response to Salim et al.'s correspondence should not have included Jeffrey Rothstein's name as an author.

SummaryProtein 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.

Introduction 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. Methods We have generated a neural stem cell line stably expressing the hDAT. Uptake kinetics of DA were determined to select a clone for transplantation. These genetically modified stem cells (or cells transfected with a construct lacking the hDAT sequence) were transplanted bilaterally into the NAC of wild-type mice trained to consume 10% alcohol in a two-bottle free-choice test for alcohol consumption. Alcohol intake was then ascertained for 1 week after transplantation, and brain sections through the NAC were examined for surviving grafted cells. Results Modified stem cells expressed hDAT and uptaken DA selectively via hDAT. Mice accustomed to drinking 10% ethanol by free choice reduced their alcohol consumption after being transplanted with hDAT-expressing stem cells. By contrast, control stem cells lacked that effect. Histologic examination revealed surviving stem cells in the NAC of all engrafted brains. Conclusions Our findings represent proof of principle suggesting that genetically engineered stem cells can be useful for exploring the role of neurotransmitters (or other signaling molecules) in alcohol consumption and potentially in other aspects of brain function.