Interactive reportRegional specification of rodent and human neurospheres
Introduction
Small populations of stem cells exist in the developing and adult rodent brain, which can generate progenitor cells capable of differentiating into neurons, astrocytes or oligodendrocytes [17]. However, fundamental challenges within this field of biology are (i) to establish how cell-autonomous programs interact with environmental signals to direct the phenotypic fate of these cells and (ii) to understand the mechanisms underlying their self-renewal capacity [2]. One technique for growing cells derived from the germinal zones of either the developing or adult CNS involves the generation of free floating spherical aggregates termed ‘neurospheres’. This method was developed for rodent tissues a number of years ago [32] and has recently been adapted for the long-term growth of human neurospheres by ourselves and others [10], [45], [49]. Neurospheres from rodents consist of both multipotent stem cells and more restricted progenitors [33] and, as such, are considered to comprise a heterogeneous population of neural precursor cells (NPCs) [41]. Although the selection of sphere forming cells from primary neurogenic zones within differentiating CNS regions of the animal is possible [34], [47], the reliable distinction between true stem cells and more restricted progenitors within expanded populations of neurospheres has been limited by the lack of available cell-type specific markers. It is possible that such cells may be regionally specified. If this were true, neurospheres generated from different brain regions would retain some features of this region, even following expansion in culture. Alternatively, is it possible that a common stem cell exists along the entire extent of the neuroaxis which, following isolation, would behave in a similar fashion irrespective of its origin. In addition to these regional complexities, there are likely to be differences between species with regard to general stem cell biology that need to be addressed, particularly in the context of potential clinical applications.
Clearly, tissues derived from one brain region and expanded in culture can take on the phenotype of another following transplantation [16], [36], [40] or, in more extreme cases, can trans-differentiate into cells of different dermal origin when injected into irradiated mice or blastocysts [5], [12]. These results suggest that at least some neuroepithelial cells are extremely plastic and environmentally specified, with very little evidence of genetic determination. However, these types of cells may be only a very small fraction of cells within such cultures. Although cells derived from different embryonic brain regions and expanded in culture adopt host region phenotypes when transplanted, there is evidence that some of these cells retain a molecular memory of where they came from, based on the expression of regionally expressed genes [28], [35], [53] or proteins [14]. In addition, although cells derived from the embryonic forebrain migrate and differentiate within hind-brain regions when transplanted into neonates [9], these cells continue to express markers associated with their region of origin [27]. Very recently, the region specific differentiation of spinal cord progenitor cells [52] and mammalian neural crest cells [51] has also been reported following transplantation. Thus, cell autonomous mechanisms may exist to control the fate of these cells. Neurospheres generated from the human fetal brain produce large number of neurons [42], but those from the spinal cord exclusively produce astrocytes using slightly different growth conditions [3], [31], suggesting that some regional specification also exists along the human neuroaxis. However, a direct comparison of proliferative and phenotypic potential of neurospheres generated from different brain regions and grown under identical culture conditions, has not previously been undertaken.
To address these issues, we compared the growth and differentiation of non-genetically modified epidermal growth factor (EGF)- and fibroblast growth factor-2 (FGF-2)-responsive neural precursors isolated from various regions of the embryonic rat and human brain.
Section snippets
Rodent neural precursor cell cultures and proliferation studies
The cortex, striatum (comprising both medial and lateral ganglionic eminences) and ventral mesencephalon (VM) were dissected from embryonic day 14 (E14) rat brain. Human embryonic tissue (between 6 and 21 weeks post conception) was collected following routine terminations of pregnancy. The methods of human tissue collection conformed with the arrangements recommended by the Polkinghorne Committee for the collection of such tissues and to the guidelines set out by the United Kingdom Department
Regional differences in growth rates of rat neurospheres
Cells derived from either the E14 rat cortex (ctxNS) or striatum (strNS) grew as neurospheres following plating and showed exponential growth over the first 35 days (Fig. 1A). These numbers represent a nearly 170-fold expansion in cell number, theoretically equivalent to approximately eight population doublings. In contrast to the forebrain neurospheres, those derived from the mesencephalon (mesNS) underwent an approximate three- to four-fold expansion (from 4×106 to 15.2±3.2×106) over the same
References (53)
Stem cells and pattern formation in the nervous system: the possible versus the actual
Neuron
(2001)- et al.
Heparin, but not other proteoglycans, potentiates the mitogenic effects of FGF-2 on mesencephalic precursor cells
Exp. Neurol.
(1998) - et al.
Regional incorporation and site-specific differentiation of striatal precursors transplanted to the embryonic forebrain ventricle
Neuron
(1995) - et al.
In vitro expansion of a multipotent population of human neural progenitor cells
Exp. Neurol.
(1999) - et al.
Subventricular zone astrocytes are neural stem cells in the adult mammalian brain
Cell
(1999) - et al.
Analysis of neural stem cells by flow cytometry: cellular differentiation modifies patterns of MHC expression
J. Neuroimmunol.
(2001) - et al.
Human neural precursor cells express low levels of telomerase in vitro and show diminishing cell proliferation with extensive axonal outgrowth following transplantation
Exp. Neurol.
(2000) - et al.
Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell
Dev. Biol.
(1996) Analysing cell lineage with a recombinant retrovirus
Trends Neurosci.
(1989)- et al.
Generation of regionally specified neurons in expanded glial cultures derived from the mouse and human lateral ganglionic eminence
Mol. Cell. Neurosci.
(2001)