Seminal Biology of Neural Stem Cells

Neurological Disorders And Stroke

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Abstract

During the embryonic period of CNS development neural stem cells (NSCs) are considered to be the proliferating cellular compartment that sustains itself through self-renewal and generates lineage-restricted progenitors, which also proliferate but ultimately differentiate into the diverse phenotypes composing later states of CNS maturation. The seminal biology of NSCs has come under intensive investigation with over 3,000 publications. However, there is still no consensus about exactly which cells are NSCs, since specific markers have not been identified. There is widespread agreement that NSCs reside in the proliferative neuroepithelium forming the core of the CNS along with their progenitor progeny. Thus, neuroepithelial cells are routinely used as a source of NSCs, and in many studies, as if they were NSCs. There is also clear agreement that more precise study of NSCs and their progeny requires identification of all the emergent phenotypes, since this would provide the opportunity to study the different stages of development and intercellular signaling mechanisms among the stages in a prospective manner. We have recently devised a novel multi-lineage labeling strategy targeting evolutionarily-conserved surface epitopes in order to identify cells progressing along neural lineages and undergoing apoptosis. We have used this strategy in conjunction with fluorescence-activated cell sorting (FACS) technology to quantify in an objective and comprehensive manner multi-lineage-negative NSCs and different lineage-positive progenitor progeny at the beginning of neurogenesis in the embryonic rat telencephalon/cortex. These FACS analyses of phenotyped cells rapidly and randomly studied with high fidelity reveal the robust stereotypical nature of cortical development and provide an initial roadmap of emergent neural phenotypes. We have then used the sort capability of the FACS technology to isolate NSCs for prospective studies into their seminal biology. In FY2005, we expanded the strategy to target nine surface epitopes in order to identify virtually all neural and non-neural phenotypes. We used the strategy with sections and dissociates of the telencephalon, which progressively develops along a ventro-dorsal gradient. FACS analyses revealed that the great majority of cells dissociated from the dorsal telencephalon were actively proliferating NSCs. Only a small minority were lineage-restricted progenitors. In contrast, FACS analyses of phenotyped cells dissociated from the ventral telencephalon demonstrated that 1) the majority were lineage-restricted neuronal and neuroglial progenitors, 2) several-fold more NSCs were apoptotic compared to those in the dorsal telencephalon, and 3) endothelial cell (ECs), which compose vessels, were 10-fold more abundant than found in the dorsal telencephalon. These results establish, for the first time, the existence of three parallel anatomical gradients in the telencephalon involving neurogenesis, apoptosis and angiogenesis. The coincident changes in cell biologies undoubtedly involve intercellular interactions among the emergent phenotypes, which remain to be elucidated. We have focused first on the roles of basic fibroblast growth factor (bFGF) and FGF receptors (FGFRs) in NSC biology. NSCs in clonal culture with bFGF exhibited four fates, which were independent of ECs and did not include EC phenotypes, thus contradicting recent publications in Science and Nature that reported studies on neuroepithelial cells. The four fates include: 1) efficient self-renewal, 2) self-renewal limited by apoptosis, 3) neurogenic only, and 4) multi-potential/phenotype. This discovery in NSC biology reflects the precision afforded by the expanded FACS strategy and clearly eclipses the existing literature. So, even among nontuple-lineage-negative NSCs there is unexpected complexity and a challenge for further study. The relative occurrence of the four fates varied with the concentration of exogenous bFGF and with the telencephalic region used as the source of NSCs. The new results show that the seminal biology of NSCs is: 1) far more complex than previously imagined, 2) determined by the relative intensity of bFGF signaling, 3) independent of ECs, and 4) itself changing noticeably along a ventro-dorsal gradient in the telencephalon. NSC fate regulation by bFGF signaling led us to investigate the roles of specific FGFRs. Multi-epitope labeling of telencephalic sections revealed widespread expressions of bFGF, FGFR1 and FGFR3 in the dorsal region and the presence of bFGF, FGFR1, FGFR2 and FGFR3 in the ventral region. Many NSCs sorted from the dorsal region co-expressed bFGF, FGFR1 and FGFR3, while many sorted from the ventral region co-expressed bFGF, FGFR1, FGFR2 and FGFR3. The roles of specific FGFRs were studied by targeting one or more FGFRs with antisense constructs. Self-renewal of NSCs from both dorsal and ventral telencephalon required the concerted activities of both FGFR1 and FGFR3, but not FGFR2. In contrast, NSC self-renewal limited by apoptosis, which was promoted by bFGF among NSCs from the ventral telencephalon, was similar under control conditions (missense constructs) and following knock-downs of both FGFR1 and FGFR3. These results suggest that activation of FGFR2, which is expressed in vivo by NSCs from the ventral, but not dorsal telencephalon, may be pro-apoptotic. Neurogenic clones, which completely predominated in vitro in the absence of added exogenous bFGF, were most effectively promoted among NSCs when all of the expressed FGFRs were knocked-down. Thus, reducing/eliminating bFGF signaling via FGFRs promoted neurogenic fates to the virtual exclusion of other developmental outcomes. Multi-potential/phenotype clones, which were strongly promoted among NSCs from the ventral but not dorsal telencephalon, were most abundant when either FGFR2 or FGFR3 alone remained active and were still present when any two FGFRs were intact. Therefore, multi-phenotype fates did not absolutely require concerted signaling through multiple FGFRs. Together, the results demonstrate that bFGF signaling levels conveyed via different FGFRs serve to determine different NSC fates in vitro. A future challenge will be to relate these results to cortical development in vivo. As a first step, we have explanted the telencephalon at E13 under control conditions and in the presence of antisense constructs targeting different FGFRs. After short-term culture, telencephalic sections and suspensions were phenotyped to assess roles of FGFRs in the context of the intact tissue. Visible changes in phenotype abundance and tissue thickness were evident that depended on the antisense treatment. Quantitative FACS analyses showed that the absolute number of NSCs was significantly reduced by knocking-down either FGFR1 or FGFR3 and significantly reduced still further by knocking-down both FGFR1 and FGFR3 or by including a pan-FGFR antagonist. These results were associated with a decrease in the percentage of proliferative NSCs, an increase in the percentage of apoptotic NSCs and a relative increase in neuronal progenitors. Thus, the results with intact explants complement those obtained from clonal analyses of sorted NSCs and further establish important roles for bFGF signaling via different FGFRs in the seminal biology of NSCs. In order to gain insight into the genes expressed by NSCs and their immediate progenitor progeny during neurogenesis in the cortex, we have collaborated with the laboratory of Lynn Hudson. NSCs and progenitors were sorted throughout neurogenesis in the cortex, and their gene expression patterns were evaluated using cDNA microanalyses. The experiments have been finished and the results are presently being analyzed.