Neural stem cells and neurospheres—re-evaluating the relationship

Abstract

For most of the past century, the prospect of replacing lost or damaged cells in the central nervous system (CNS) was hampered by the opinion that the adult mammalian CNS was incapable of generating new nerve cells. This belief, like most dogmas, was essentially founded on a lack of experimental evidence to the contrary. The overturning of this 'no new neuron' hypothesis began midway through the twentieth century with a series of reports documenting neurogenesis in the postnatal and adult brain, continued with the isolation and in vitro culture of neurogenic cells from the adult mammalian brain, and culminated in the discovery of a population of multipotent, self-renewing cells in the adult CNS (that is, bona fide neural stem cells). Although a variety of techniques were initially used, the neurosphere assay (NSA) rapidly emerged as the assay of choice and has since become a valuable tool for isolating, and understanding the biology of, embryonic and adult CNS stem cells. Like all technologies, it is not without its limitations. In this article we will highlight several shortcomings of the assay related to its application and interpretation that we believe have led to a significant body of research whose conclusions may well be misleading.

The culture of neural stem cells

“…Can some NPs also form NS and how do we distinguish which cells, NSCs or NPs, are giving rise to NS. Reynolds and Rietze [2005], in their interesting perspective, suggest that growth rates of cells in NS culture can be used to test the idea of whether NFUs truly reflect the number of NSCs in any particular culture. Their starting point is to say that clonal NFU analysis suggest that there is 2.4% NSCs in NS cultures.…”

“…A further complication of the analysis of Reynolds and Rietze [2005] is that in NS bulk culture at high density there is significant NS aggregation [Singec et al, 2006;Mori et al, 2007;Coles-Takabe et al, 2008]. This phenomenon has been observed most directly by differential marking of cells (with actin promoter driven EYFP and dsRed) followed by NS color analysis [Coles-Takabe et al, 2008].…”

Stage- and area-specific control of stem cells in the developing nervous system

“…Therefore, it is likely that other TGF superfamily members, signaling via a different type II receptor, compensate for the loss of TGF signaling. Proof for intrinsic differences comes from in vitro neurosphere [40] experiments, in which self-renewal of midbrain, but not forebrain NSCs was reduced by the addition of TGF1. This differential responsiveness likely involves the transcription factor FoxG1 that is specifically expressed in the forebrain neuroepithelium.…”

ABC transporters, neural stem cells and neurogenesis – a different perspective

“…Neurospheres are useful to evaluate NSC multipotentiality (through the character- [40]. Neurospheres are floating structures that can be obtained by exposing dissociated embryonic or adult CNS tissue to growth factors [41][42][43]. These heterogeneous spheroid structures contain NSCs/progenitors and differentiated cells embedded in a complex extracellular matrix and organized three dimensionally (3D) with a core of differentiating GFAP + and b-tubulin III + cells surrounded by nestin+, epidermal growth factor receptor (EGFR) + and b1-integrin + undifferentiated cells [40].…”

“…Histotypical 3D neurospheres in suspension culture are thus characterized as heterogeneous clusters containing unequal stem cell subtypes where nestin + dividing cells surround a core of differentiated GFAP + and b-tubulin III + cells. Cell divisions can be found mainly at the edge of the neurosphere where NSC markers such as notch1 and nestin are also expressed [41]. This pattern suggests that NSCs may be a rare subgroup of nestin + cells that simultaneously express a combination of markers, such as LeX/ssea1/b1-integrin/notch1 [40,41,45].…”