Eric D. Laywell

Human cortical glial tumors contain neural stem-like cells expressing astroglial and neuronal markers in vitro

Neural stem cells from neurogenic regions of mammalian CNS are clonogenic in an in vitro culture system exploiting serum and anchorage withdrawal in medium supplemented with methyl cellulose and the pleiotropic growth factors EGF, FGF2, and insulin. The aim of this study was to test whether cortical glial tumors contain stem‐like cells capable, under this culture system, of forming clones showing intraclonal heterogeneity in the expression of neural lineage‐specific proteins. The high frequencies of clone‐forming cells (about 0.1–10 × 10−3) in clinical tumor specimens with mutated p53, and in neurogenic regions of normal human CNS, suggest that the ability to form clones in this culture system is induced epigenetically. RT‐PCR analyses of populations of normal brain‐ and tumor‐derived sister clones revealed transcripts for nestin, neuron‐specific enolase, and glial fibrillary acidic protein (GFAP). However, the tumor‐derived clones were different from clones derived from neurogenic regions of normal brain in the expression of transcripts specific for genes associated with neural cell fate determination via the Notch‐signaling pathway (Delta and Jagged), and cell survival at G2 or mitotic phases (Survivin). Moreover, the individual glioma‐derived clones contain cells immunopositive separately for GFAP or neuronal β‐III tubulin, as well as single cells coexpressing both glial and neuronal markers. The data suggest that the latent critical stem cell characteristics can be epigenetically induced by growth conditions not only in cells from neurogenic regions of normal CNS but also in cells from cortical glial tumors. Moreover, tumor stem‐like cells with genetically defective responses to epigenetic stimuli may contribute to gliomagenesis and the developmental pathological heterogeneity of glial tumors. GLIA 39:193–206, 2002.

MESENCHYMAL STEM CELLS SPONTANEOUSLY EXPRESS NEURAL PROTEINS IN CULTURE AND ARE NEUROGENIC AFTER TRANSPLANTATION

Reports of neural transdifferentiation of mesenchymal stem cells (MSCs) suggest the possibility that these cells may serve as a source for stem cell–based regenerative medicine to treat neurological disorders. However, some recent studies controvert previous reports of MSC neurogenecity. In the current study, we evaluate the neural differentiation potential of mouse bone marrow–derived MSCs. Surprisingly, we found that MSCs spontaneously express certain neuronal phenotype markers in culture, in the absence of specialized induction reagents. A previously published neural induction protocol that elevates cytoplasmic cyclic AMP does not upregulate neuron‐specific protein expression significantly in MSCs but does significantly increase expression of the astrocyte‐specific glial fibrillary acidic protein. Finally, when grafted into the lateral ventricles of neonatal mouse brain, MSCs migrate extensively and differentiate into olfactory bulb granule cells and periventricular astrocytes, without evidence of cell fusion. These results indicate that MSCs may be “primed” toward a neural fate by the constitutive expression of neuronal antigens and that they seem to respond with an appropriate neural pattern of differentiation when exposed to the environment of the developing brain.

Microglia Instruct Subventricular Zone Neurogenesis

Microglia are increasingly implicated as a source of non‐neural regulation of postnatal neurogenesis and neuronal development. To evaluate better the contributions of microglia to neural stem cells (NSCs) of the subventricular neuraxis, we employed an adherent culture system that models the continuing proliferation and differentiation of the dissociated neuropoietic subventricular tissues. In this model, neuropoietic cells retain the ability to self‐renew and form multipotent neurospheres, but progressively lose the ability to generate committed neuroblasts with continued culture. Neurogenesis in highly expanded NSCs can be rescued by coculture with microglial cells or microglia‐conditioned medium, indicating that microglia provide secreted factor(s) essential for neurogenesis, but not NSC maintenance, self‐renewal, or propagation. Our findings suggest an instructive role for microglial cells in contributing to postnatal neurogenesis in the largest neurogenic niche of the mammalian brain.

Cerebellar Disorganization Characteristic of Reeler in Scrambler Mutant Mice Despite Presence of Reelin

Analysis of the molecular basis of neuronal migration in the mammalian CNS relies critically on the discovery and identification of genetic mutations that affect this process. Here, we report the detailed cerebellar phenotype caused by a new autosomal recessive neurological mouse mutation, scrambler (gene symbolscm). The scrambler mutation results in ataxic mice that exhibit several neuroanatomic defects reminiscent of reeler. The most obvious of these lies in the cerebellum, which is small and lacks foliation. Granule cells, although normally placed in an internal granule cell layer, are greatly reduced in number (∼20% of normal). Purkinje cells are also reduced in number, and the majority are located ectopically in deep cerebellar masses. There is a small population of Purkinje cells (∼5% of the total) that occupy a Purkinje cell layer between the molecular and granule cell layers. Despite this apparent disorganization of Purkinje cells, zebrin-positive and zebrin-negative parasagittal zones can be delineated. The ectopic masses of Purkinje cells are bordered by the extracellular matrix protein tenascin and by processes containing glial fibrillary acidic protein. Antibodies specific for these proteins also identify a novel midline raphe structure in both scrambler and reeler cerebellum that is not present in wild-type mice. Thus, in many respects, the scrambler cerebellum is identical to that of reeler. However, the scrambler locus has been mapped to a site distinct from that of reelin (Reln), the gene responsible for the reeler defect. Here we find that there are normal levels of Reln mRNA in scrambler brain and that reelin protein is secreted normally by scrambler cerebellar cells. These findings imply that the scrambler gene product may function in a molecular pathway critical for neuronal migration that is tightly linked to, but downstream of, reelin.

A nestin-negative precursor cell from the adult mouse brain gives rise to neurons and glia

Using a novel suspension culture approach, previously undescribed populations of neural precursor cells have been isolated from the adult mouse brain. Recent studies have shown that neuronal and glial precursor cells proliferate within the subependymal zone of the lateral ventricle throughout life, and a persistent expression of developmentally regulated surface and extracellular matrix molecules implicates cell‐cell and cell‐substrate interactions in the proliferation, migration, and differentiation of these cells. By using reagents that may affect cell‐cell interactions, dissociated adult brain yields two types of cell aggregates, type I and type II spheres. Both sphere types are proliferative, and type I spheres evolve into type II spheres. Neurons and glia arise from presumptive stem cells of type II spheres, and they can survive transplantation to the adult brain. GLIA 21:399–407, 1997.

Multipotent Neurospheres Can Be Derived from Forebrain Subependymal Zone and Spinal Cord of Adult Mice after Protracted Postmortem Intervals

The adult mammalian CNS harbors a population of multipotent stem/progenitor cells that can be induced to grow as proliferative neurospheres in vitro. We demonstrate here that neurosphere-generating cells can be isolated from adult mouse spinal cord and forebrain subependymal zone after postmortem intervals of up to 140 h, when kept at 4 degrees C, and up to 30 h when kept at room temperature. Although there was an inverse relationship between postmortem interval and the number of neurospheres generated, neurospheres derived under these conditions were proliferative and could give rise to both neurons and glia.

Extracellular matrix effects on neurosphere cell motility.

There is a paucity of information on the roles of extracellular matrix (ECM) and substrate molecules in general with regard to the growth and differentiation of neural stem and progenitor cells. There are well-established findings of a dense, presumably astrocyte-derived ECM in the persistently neurogenic subependymal zone and its migratory extension the rostral migratory stream. Cells cultured from this region, as well as from early postnatal cerebellum, generate multipotent neurospheres, but at present there is little information as to the ECM regulation of these neural stem cell populations. The present study examined the behavior of cerebellar-derived neurospheres on the matrix components laminin, fibronectin, and chondroitin sulfate proteoglycan. The results showed that laminin and fibronectin significantly increase cell migration velocity as compared to CSPG. Fibronectin effected a maximal velocity after 48 h, whereas maximal velocity on laminin and CSPG was not reached until 72 h. Both laminin and fibronectin were very permissive substrates for cellular outgrowth. Chondroitin sulfate proteoglcyan showed a significant inhibition of migratory outgrowth and velocity. These ECM molecules did not appear to affect the fate choice of neurons and glia, thus their role in neuropoietic structures may be to facilitate or deter cell movement and process outgrowth.

Boundaries and wounds, glia and glycoconjugates. Cellular and molecular analyses of developmental partitions and adult brain lesions.

During brain development, transient partitions of glia and glycoconjugates (glycoproteins, glycolipids, and glycosaminoglycans) surround forming functional units (e.g., nuclear divisions, whisker-related barrels, and neostriatal striosomes). These partitions, which we think of as boundaries, consist of dense aggregates of glial fibrillary acidic protein (GFAP)-positive radial glia, young astrocytes and their processes, and developmentally regulated glycoconjugates (e.g., J1/tenascin and the 473 proteoglycan) that can be thought of as recognition molecules present on membranes or perhaps within the extracellular matrix. When functional patterns have formed and appear to be stabilized, these boundaries are no longer detectable. Lesions of the developing brain show the existence of a more global astrocytic distribution suggestive of biochemically distinct subsets of astrocytes that reside within boundary versus nonboundary positions. Lesions of the adult brain, in addition to showing gliosis, reveal a reexpression of some of the same macromolecules present in transient brain boundaries during development. It is postulated that developmental boundaries and wounds in the adult brain possess some of the same inhibitory and possibly alluring molecular substrates for neuritic expansion.

Astrocytes as stem cells: Nomenclature, phenotype, and translation

Recently discovered multipotent astrocytic stem cells are discussed in light of current nomenclature for glial precursor and lineage‐associated cells in the developing, postnatal, and adult mammalian brain. Defining the phenotype of any immature cell in the nervous system is a challenge, and a position is stated that includes the need for categorizing cells within a continuum of differentiation potential. The possibility for dedifferentiating glial cells into clonogenic stem‐like cells offers numerous possibilities for translating knowledge and technology from this subfield of stem cell biology to regenerative medicine. Along with the need for developing a new lexicon for defining the cellular players that contribute to the generation of glia and neurons in the developing and mature central nervous system, the relationships also need to be established among potency, repopulation attempts, and tumorigenesis of cells meeting the criteria of glial stem cells. Finally, it is possible that understanding the normal differentiation, de‐ and transdifferentiation potential of glial stem‐like cells in the mature central nervous system will provide insights into the possible use of these cells, or biogenic factors associated with their growth and differentiation, in therapeutic approaches for a variety of neurological disorders. GLIA 43:62–69, 2003.

The Complex Nature of Interactive Neuroregeneration-Related Molecules

We review the growing list of molecules that may be involved in wound healing in the central nervous system (CNS). It is known that many of these molecules are present during normal development and neoplastic growth in both neural and nonneural tissues, often in areas where pattern formation or tissue remodeling is evident; however, their functional roles are often quite elusive. In order to understand the changes that occur in and around a brain wound, we review proposed functions of neuroregeneration-related molecules in in vitro and in vivo preparations, as well as note their expression in other healing tissues including skin. A hypothesis that wound healing events in the CNS supersede neuritic growth around a lesion is presented. In contrast to the classical view of failed regeneration, there may be significant amounts of circuit reorganization that occur following injury, and such plasticity may be further enhanced by manipulating the molecular environment around a brain wound and in synaptically related structures.