Valery G. Kukekov

Neural stem cell heterogeneity demonstrated by molecular phenotyping of clonal neurospheres

Neural stem cells (NSCs) in vitro are able to generate clonal structures, “neurospheres,” that exhibit intra-clonal neural cell-lineage diversity; i.e., they contain, in addition to NSCs, neuronal and glial progenitors in different states of differentiation. The present study focuses on a subset of neurospheres derived from fresh clinical specimens of human brain by using an in vitro system that relies on particular growth factors, serum, and anchorage withdrawal. Thirty individual and exemplary cDNA libraries from these neurosphere clones were clustered and rearranged within a panel after characterization of differentially expressed transcripts. The molecular phenotypes that were obtained indicate that clonogenic NSCs in our in vitro system are heterogeneous, with subsets reflecting distinct neural developmental commitments. This approach is useful for the sorting and expansion of NSCs and facilitates the discovery of genes involved in cell proliferation, communication, fate control, and differentiation.

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.

Chapter 15 Boundary molecules during brain development, injury, and persistent neurogenesis - in vivo and in vitro studies

Publisher Summary In this chapter, three model systems have been described that exemplify the presence and potential biological functions of boundary extracellular matrix (ECM) molecules. Even though these developmentally regulated proteins, sometimes referred to as recognition molecules, are often uniformly expressed in low-levels throughout the developing neuraxis, their dense concentration in so-called boundary regions alerted to their possible functions as interfaces between different brain structures or units. This cordone hypothesis is also consistent with the observed upregulation of boundary molecules in association with the glial scar, and bioassays of such molecules in the astroglial scar reveal their complex interactions with numerous other molecules that can both encourage or deter neuronal adhesion and neurite growth. The persistent expression of boundary ECM molecules in the neurogenic subependymal zone (SEZ) may prove to be the most insightful model for studying the many roles of these molecules. Because cells are born, migrate, and die in relation to an enhanced ECM expression in vivo in the rostral migratory stream, as well as the inferred role of ECM in the generation of novel proliferative spheres in vitro , future studies need to focus on the patterns of gene expression of SEZ-derived stem and precursor cells in relation to ECM expression in order to establish their roles during the proliferation and specification of neurons and glia.

Neural Stem/Progenitor Cell Clones or “Neurospheres”

Stem cell biology has contributed an impressive list of new and important findings in the last decade that are anticipated to lead to potentially powerful therapeutics for debilitating human diseases. The generation of human embryonic stem cell (ES cell) lines (1,2) is among this list of crucial technological breakthroughs, not to mention the applications of genetic, cell, and molecular biology to the first-time cloning of an entire organism (3) that has since been achieved in numerous species, including the mouse (4). Utilizing insights and approaches from these fields, as well as from developmental biology, the field of developmental neurobiology has been astonished in recent years by numerous “reversals of dogma” related to neurogenesis in the mature mammalian brain [defined as the generation of neurons, or the shortened form of “neuronogenesis,” versus “gliogenesis,” which is the production of astroglia and oligodendroglia; “neuromorphogenesis” is the combined events of neurogenesis and gliogenesis that ultimately generate a nervous system (5–8)]. In particular, despite the work of Allen (9), Altman and Das (10),and others supporting the existence of persistent neurogenesis in the adult rat olfactory and hippocampal systems, these were considered highly specialized cases that by no means supported a notion of neuropoiesis (persistent neurogenesis) in the adult central nervous system. The in vitro propagation of a putative stem cell population from the adult rat brain by the Weiss and Bartlett groups (11,12) suggested that there may be neuropoiesis; studies that followed have established the source of these stem/progenitor cells [a term used because it not only sidesteps the contentious issue of sternness (5,8,13) of these cells, but also because it encompasses the entire spectrum of proliferative neurogenic cells that can generate all cells in the nervous system] as the subependymal zone, ependyma, and hippocampus, even within the aged human brain (6, 14–16). It is possible to clone stem/progenitor cells from these adult brain regions (7) and even from cadaver specimens (17,18) with surprisingly long postmortem intervals [up to 5 d from cadaver specimens (17)]. For the purpose of discussion, these persistently neurogenic regions have been amalgamated under one term—“brain marrow” (8, 14,19). The analogy of a brain neuropoietic core to the hematopoietic bone marrow has been substantiated by the recent surprising finding that adult brain-derived stem/progenitor cells are considerably more pluripotent than ever expected [e.g., giving rise to blood cells after homing to bone marrow following systemic grafting (20), as well as to muscle (21,22) and even multiple organ systems (23)]; it also has recently been shown that non-neural stem cells can also be coaxed into nerve cell phenotypes following different neuralizing conditions (24–27). However, to date, most transplant or other in vivo studies of stem/progenitor cells derived from brain marrow suggest that these cells have a rather limited fate potential in the central nervous system (CNS) [e.g., glia (28)]—but also giving rise to granule cells and other interneuron populations although fetal neural stem cells and immortalized neural stem cell lines do appear to be more plastic and potent, incorporating into a variety of brain circuits (29–31).