Neural Stem Cells and their Therapeutic Potenial

Introduction

In contrast to previous opinions, it is now well established that neurogenesis occurs in the adult mammalian brain, at least in restricted areas where cells with stem cell like properties can be found. While great efforts have been expended to investigate the intrinsic properties of neural stem cells (NSCs) and the factors regulating their differentiation during the last decade, recent lines of research have begun to explore their therapeutic potential. This essay briefly summarizes the current state of stem cell research and gives a survey of the experimental approaches employed to investigate potential therapeutic applications of NSCs in the treatment of Parkinson disease and glioblastoma.

Neural stem cells in the adult central nervous system

History

It has long been thought that differentiation and regeneration in the central nervous system (CNS) would only be possible during development. “Once the development was ended, the fonts of growth and regeneration …. dried up irrevocably”, as Ramon y Cajal (1928) summarized the current opinion on this issue in the early 20th century. Cajal´s statement was based on the observation that there was no apparent mitotic activity in CNS, and that intermediate forms which could be considered to represent early forms of the high complex neurons seemed to be lacking. This view was challenged by the introduction of the 3H-thymidine autoradiographic method which allowed for the identification of dividing cells^[1]. By using this technique, Altman (1962) was the first to propose neurogenesis in the mammalian brain, and in a series of papers published during the 1960s he reported the existence of newly formed neurons in various brain regions , including the olfactory bulb, the hippocampus, and the neocortex (see Gross, 2000, for a review). The occurrence of neurogenesis in the adult CNS was subsequently established by the introduction of the thymidine analogue BrdU¹ as another in vivo marker of proliferating cells which can be combined with immunostaining for cell-type specific markers (Lois and Alvarez-Buylla 1993) and by the establishment of a culture method for NSCs (Reynolds and Weiss 1992).

Characteristics and characterization of neural stem cells

Somatic SC (in contrast to totipotent embryonic SC) are commonly characterized by the following three features: they are mitotically competent, self-renewing, and multipotent cells (Gage 1998; Okano 2002b; Okano 2002a; Villa et al. 2002). In case of NSCs, the term multipotency characterizes the ability to differentiate along the three main cell lineages in the CNS: neurons, astrocytes, and oligodendrocytes. Since it is not fully elucidated yet whether NSCs are able to meet the ultimate criterion for stem cells, i.e., to reconstitute the entire organ they are derived from, Snyder (1997) suggested to apply the term “stem-like cells” to self-replicating, multipotent cells in the CNS.

First evidence for the existence of a cell type with multilineage potential and self-replicating capacity in the adult CNS was provided by the neurosphere formation assay developed by Reynold & Weiss (1992, see Fig.1). However, this novel culture method only permits a retrospective identification of NSCs, i.e. only after a neurosphere is formed, it is possible to conclude whether the initial cell was a NSCS. Similarly, recently identified marker molecules for NSCs, such as Musashi1 (an RNA-binding protein), Nestin (an intermediate filament), and Sox1 (a transcription factor), cannot be used for the prospective identification of living of NSCs by means of immunoassaying since these molecules are no cell-surface antigens, as it is the case, for instance , with the specific marker molecules of haematopoietic stem cells.

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Recently, Kawaguchi et al. (Kawaguchi et al. 2001) reported an exciting approach for a prospective identification of NSCs, which is based on the construction of a life reporter gene in which enhanced green fluorescent protein (EGFP) is expressed under the transcriptional control of the second intronic enhancer for the gene encoding Nestin, which acts highly selective for NSCs, allowing for the isolation of NSCs by fluorescence- activated cell sorting (FACS).

Localization of neural stem cells in the mammalian brain

Until today, two neurogenic sites have been identified in the adult mammalian brain, the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone of the hippocampal dentate gyrus (DG).

Based on their neurosphere formation assay, Reynolds and Weiss (1992) firstly cultured multipotent NSCs from the adult rat striatum, though it was consecutively shown that these cells originated in the SVZ (Morshead et al. 1994). The generation of new neurons in the SVZ, possibly by ependymal cells (Johansson et al. 1999), serve to replace interneurons in the olfactory bulb, and neuronal cells move all the way to the olfactory bulb through a pathway called the rostral migratory pathway (Doetsch and Alvarez Buylla 1996). Similarly, neurons are generated from progenitors in the subgranular zone of the dentate gyrus and migrate to the granule cell layer where they differentiate into neurons. (Reviewed in Gould and Gross 2002).

The existence of NSCs has also been reported at non-neurogenic sites. Weiss et al. ((Weiss et al. 1996) isolated cells from murine spinal cord, a site where normally no neurogenesis occurs, which were then induced to proliferate and differentiate into neurons or glial cells, depending on the environment. Moreover, NSCs were isolated from the septum and striatum of rats (Palmer et al. 1995). These findings indicate that the restriction of neurogenesis to discrete brain areas might be due to local differences in the signalling environment rather than lack of NSCs at respective brain sites.

Functions of neural stem cells in the adult brain

The hippocampus is widely believed to be involved in spatial forms of learning and memory (Morris et al. 1982). Recent studies indicate that the extend of neurogenesis in the dentate gyrus is correlated to learning processes. Kempermann et al. (Kempermann et al. 1997) found that mice exposed to an enriched environment showed a substantial increase in the proliferation rate of DG neurons as compared to controls and performed significantly better learning task paradigms. Moreover, Beylin et al. (2001) reported that rats with artificially reduced hippocampal neurogenesis have performed worse than controls in hippocampus-dependent tasks assessing associative learning, while task performance remained unimpaired in a hippocampus-independent task paradigm. Although further investigation will be necessary to elucidate the exact causal relationship between learning and neurogenesis in the DG, it can be concluded that adult neurogenesis may be affected by experience, which is learning in a very broad sense, and that alterations in the neurogenic activity has a clear effect on behaviour.

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^[1] Radioactive 3H-thymidine and the thymidine analogue bromodeoxyuridine (BrdU) can be introduced into proliferative cells which in turn incorporate them into the DNA during the S-phase of the cell cycle, prior to cell division. Recording the radioactive signal emitted by the incorporated 3H-thymidine or applying monoclonal antibodies for BrdU allow to trace the progeny of dividing cells in the respective methods.