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Regional specification of rodent and human neurospheres

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Abstract

Neural precursor cells were isolated from various regions of the developing rat and human brain and grown in culture as aggregates termed neurospheres. We asked whether cells within human and rodent neurospheres are identical, or whether they have species specific characteristics or differences based on their region of origin. Under our culture conditions, rodent neurospheres isolated from the cortex (ctxNS) and striatum (strNS) grew faster than those from the mesencephalon (mesNS), but stopped growing after only eight to ten population doublings. In contrast, human neurospheres under identical culture conditions, continued to grow for over 40 population doublings. Following migration and differentiation of both rodent and human cultures, ctxNS and strNS generated high numbers of small neurons whereas mesNS generated small numbers of large neurons with many long fibres. Only very rare neurons from mesNS expressed dopaminergic markers, and thus may require further signals to fully mature. While the rat neurospheres generated high numbers of oligodendrocytes, very few were found to develop from human neurospheres from any region after a few weeks of passaging. FACS analysis revealed a unique population of smaller cells within human strNS and ctxNS, which appeared to be neuronal progenitors. However, large cells within neurospheres were capable of generating these small neuronal progenitors following further proliferation. Together, our data show that rat and human neurospheres have unique characteristics with regard to growth and differentiation, and that the majority of precursor cells within neurospheres are regionally specified to generate set numbers of neurons. These findings have important implications for understanding the nature of proliferating neural precursors isolated from the developing CNS, and their potential for brain repair.

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

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