Human neurospheres derived from the fetal central nervous system are regionally and temporally specified but are not committed

Abstract

Proliferating single cells were isolated from various CNS regions (telencephalon, diencephalon, midbrain, cerebellum, pons and medulla, and spinal cord) of human fetal cadavers at 13 weeks of gestation and grown as neurospheres in long-term cultures. We investigated whether neural stem cells (NSCs) or progenitors within spheres have specific regional or temporal characteristics with regard to growth, differentiation, and region-specific gene expression, and whether these molecular specifications are reversible. Regardless of regional origin, all of the neurospheres were found to contain cells of different subtypes, which suggests that multipotent NSCs, progenitors or radial glial cells co-exist with restricted neuronal or glial progenitors within the neurospheres. Neurospheres from the forebrain grew faster and gave rise to significantly more neurons than did those from either the midbrain or hindbrain, and regional differences in neuronal differentiation appeared to be sustained during long-term passage of neurospheres in culture. There was also a trend towards a reduction in neuronal emergence from the respective neurospheres over time in culture, although the percentages of neurons generated from cerebellum-derived neurospheres increased dramatically. These results suggest that differences in neuronal differentiation for the various neurospheres are spatially and temporally determined. In addition, the properties of glial fibrillary acidic protein (GFAP)-, glutamate-, and γ-aminobutyric acid (GABA)-expressing cells derived from neurospheres of the respective CNS regions appear to be regionally and temporally different. Isolated human neurospheres from different CNS compartments expressed distinctive molecular markers of regional identity and maintained these patterns of region-specific gene expression during long-term passage in vitro. To determine the potential of human neurospheres for regional fate plasticity, single spheres from the respective regions were co-cultured with embryonic day 16.5 (E16.5 d) mouse brain slices. Specific cues from the developing mouse brain tissues induced the human neurospheres to express different marker genes of regional identity and to suppress the expression of their original marker genes. Thus, even the early regional identities of human neurospheres may not be irreversible and may be altered by local inductive cues. These findings have important implications for understanding the characteristics of growth, differentiation, and molecular specification of human neurospheres derived from the developing CNS, as well as the therapeutic potential for neural repair.

Introduction

Multipotent neural stem cells (NSCs) or progenitors derived from the developing or adult central nervous system (CNS) can be isolated in culture and propagated in the presence of mitogens (for review, see Gage, 2000). One technique for growing these cells involves the generation of free-floating spherical aggregates, which are termed ‘neurospheres’. This method was developed a number of years ago for rodent tissues (Reynolds and Weiss, 1992). Subsequently, the technique was adapted for generating human neurospheres from postmortem fetal CNS tissues, using a variety of growth factors (Svendsen et al., 1997, Flax et al., 1998, Carpenter et al., 1999, Vescovi et al., 1999, Ostenfeld et al., 2000, Caldwell et al., 2001). Human neurospheres have great potential for cell therapy applications and as a model for studying human development and disease (Flax et al., 1998, Fricker et al., 1999, Vescovi et al., 1999, Ostenfeld et al., 2000, Englund et al., 2002, Park et al., 2002a).

Neurospheres from rodents or humans consist of both multipotent stem cells and more restricted progenitors at different stages of differentiation and, as such, are considered to comprise a heterogeneous population of neural precursor cells (Reynolds and Weiss, 1996, Svendsen and Caldwell, 2000, Suslov et al., 2002). Oligonucleotide microarray technology, which facilitates large-scale screening of gene expression in stem cells from a wide range of tissues, has been used to show that isolated human NSC populations are heterogeneous and have variable patterns of gene expression (Suslov et al., 2002). In addition to the cellular heterogeneity that occurs within neurospheres, there is evidence that rodent and human neurospheres are regionally specified. Neurospheres isolated from different regions of the developing or adult brain display unique characteristics with regard to growth, differentiation, and gene expression (Hitoshi et al., 2002, Ostenfeld et al., 2002, Horiguchi et al., 2004).

Regional patterning in the developing mammalian brain is partially regulated by restrictive region-specific regulatory gene expression patterns within the germinal zone, which is composed of stem cells and their progenitor cell progenies. Thus, NSCs, together with their progeny, are poised to receive inductive signals that establish regional identity in vivo. Mouse neurospheres isolated from different regions of the brain display region-specific gene expression patterns, although commitment to this regional identity is not absolute and can be altered by local inductive cues (Hitoshi et al., 2002, Santa-Olalla et al., 2003). Furthermore, timing seems to be encoded in the NSCs or progenitors, which means that they have positional information as well as temporal information, manifested as stage-dependent changes in the NSCs or progenitors (Temple, 2001). The neurospheres derived from the rat brain produce progressively fewer neurons, but the number of glial cells increases with the number of passages in vitro (Ostenfeld et al., 2002). Finally, there are significant differences in neurosphere growth patterns between the rat, mouse, and humans (Ginis and Rao, 2003, Snyder et al., 2004). However, the temporal patterns of growth, differentiation, and gene expression in these cultures and the precise cellular composition of human neurospheres have not been fully clarified.

In this study, NSCs or progenitors were isolated from the respective regions of the developing human CNS and grown in long-term cultures as neurospheres. We investigated whether NSCs or progenitors within neurospheres have specific regional and/or temporal characteristics with regard to growth, differentiation, and gene expression during expansion in culture and whether the characteristics of gene expression can be reversed by local environmental factors.