Segregation of human neural stem cells in the developing primate forebrain.

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.

Transplantation of neural progenitor and stem cells: developmental insights may suggest new therapies for spinal cord and other CNS dysfunction.

Multipotent neural progenitors and stem cells may integrate appropriately into the developing and degenerating central nervous system. They may also be effective in the replacement of genes, cells, and nondiffusible factors in either a widespread or a more circumscribed manner, depending on the therapeutic demands of the clinical situation. In addition, they may be uniquely responsive to some types of neurodegenerative conditions. We believe that these various appealing capabilities are the normal expression of basic biologic properties and attributes of a stem cell. The therapeutic utility of some of those properties is illustrated in this review of ongoing work in our laboratory, particularly with regard to spinal dysfunction. In these examples, we believe we have tapped into a mechanism that underlies a remarkable degree of natural plasticity programmed into the nervous system at the cellular level, and we have now exploited those properties for therapeutic ends.

Sonic hedgehog regulates proliferation and inhibits differentiation of CNS precursor cells.

Activation of the Sonic hedgehog (Shh) signal transduction pathway is essential for normal pattern formation and cellular differentiation in the developing CNS. However, it is also thought to be etiological in primitive neuroectodermal tumors. We adapted GAL4/UAS methodology to ectopically express full-length Shh in the dorsal neural tube of transgenic mouse embryos commencing at 10 d postcoitum (dpc), beyond the period of primary dorsal-ventral pattern formation and floorplate induction. Expression of Shh was maintained until birth, permitting us to investigate effects of ongoing exposure to Shh on CNS precursors in vivo. Proliferative rates of spinal cord precursors were twice that of wild-type littermates at 12.5 dpc. In contrast, at late fetal stages (18.5 dpc), cells that were Shh-responsive but postmitotic were present in persistent structures reminiscent of the ventricular zone germinal matrix. This tissue remained blocked in an undifferentiated state. These results indicate that cellular competence restricts the proliferative response to Shh in vivo and provide evidence that proliferation and differentiation can be regulated separately in precursor cells of the spinal cord. Thus, Hedgehog signaling may contribute to CNS tumorigenesis by directly enhancing proliferation and preventing neural differentiation in selected precursor cells.

Engraftable human neural stem cells respond to developmental cues, replace neurons, and express foreign genes.

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.

Multipotent neural precursors can differentiate toward replacement of neurons undergoing targeted apoptotic degeneration in adult mouse neocortex.

Neurons undergoing targeted photolytic cell death degenerate by apoptosis. Clonal, multipotent neural precursor cells were transplanted into regions of adult mouse neocortex undergoing selective degeneration of layer II/III pyramidal neurons via targeted photolysis. These precursors integrated into the regions of selective neuronal death; 15 +/- 7% differentiated into neurons with many characteristics of the degenerated pyramidal neurons. They extended axons and dendrites and established afferent synaptic contacts. In intact and kainic acid-lesioned control adult neocortex, transplanted precursors differentiated exclusively into glia. These results suggest that the microenvironmental alterations produced by this synchronous apoptotic neuronal degeneration in adult neocortex induced multipotent neural precursors to undergo neuronal differentiation which ordinarily occurs only during embryonic corticogenesis. Studying the effects of this defined microenvironmental perturbation on the differentiation of clonal neural precursors may facilitate identification of factors involved in commitment and differentiation during normal development. Because photolytic degeneration simulates some mechanisms underlying apoptotic neurodegenerative diseases, these results also suggest the possibility of neural precursor transplantation as a potential cell replacement or molecular support therapy for some diseases of neocortex, even in the adult.

Expression of human beta-hexosaminidase alpha-subunit gene (the gene defect of Tay-Sachs disease) in mouse brains upon engraftment of transduced progenitor cells.

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.

Myelination in the developing human brain: biochemical correlates.

To delineate the biochemical sequences of myelination in the human brain, we analyzed the protein and lipid composition of white matter in 18 baseline cases ranging in age from midgestation through infancy, the critical period in human myelination when the most rapid changes occur. Three adult cases were used as indices of maturity, and 4 cases with major disorders of CNS myelination (maple syrup urine disease, severe periventricular leukomalacia, idiopathic central hypomyelination, and metachromatic leukodystrophy) were analyzed. Brain samples were obtained < or = 24 hours after death. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high performance thin-layer chromatography were used to separate and identify proteins and polar and neutral lipids in an average of 10 sites/brain; computer-based densitometry was used to quantify polar lipids. Biochemical sequences, as manifested by the appearance of the myelin-associated lipids and myelin-specific proteins, closely followed previously described anatomic sequences both temporally and by region, and were identical in all sites sampled: sphingomyelin was followed simultaneously by cerebrosides, MBP, PLP, and nonhydroxy-sulfatide, followed by hydroxy-sulfatide. The onset and tempo of the expression of individual constituents, however, were quite variable among sites, suggesting a wide differential in vulnerable periods to insult in biochemically-specific pathways in early life. Cholesterol ester was transiently elevated during late gestation and early infancy, prior to and around the time of the appearance of cerebrosides, sulfatides, PLP, and MBP. Distinctive lipid and protein abnormalities were detected in idiopathic central hypomyelination and metachromatic leukodystrophy. This study underscores the feasibility of the combined biochemical approaches in pediatric brains and provides guidelines for the assessment of disorders of myelination in early human life.