During CNS development NSCs are considered to be the cellular compartment that both expands through self-renewal and generates lineage-restricted progenitors. Progenitors ultimately differentiate into the neural phenotypes composing different stages of CNS development. Although the seminal biology of NSCs has come under intensive investigation, there is still no consensus regarding exactly which cells are actually NSCs, since specific markers do not exist. So, neuroepithelial cells are widely used as the primary source of NSCs, and often as if they all were NSCs. And, NSCs are widely identified functionally as cells that can both self-renew and differentiate into multiple neural phenotypes. Thus, there is a general consensus that further elucidation of NSC biology first requires their phenotypic identification since this could provide direct experimental access to them for prospective rather than retrospective investigation. During FY2009 we focused entirely on relating our phenotyping strategy to all of the available markers currently used in the field. Different investigators use different markers and methods to try to identify putative NSCs from retrospective analyses of heterogeneous cell populations exhibiting the generic functional endpoints of putative NSC phenotypes (self-renewal and multipotent differentiation). The results have led to a continuing lack of consensus regarding the seminal properties of true NSCs and their lineal relationships. So, many putative NSC phenotypes are simply lumped together, since investigators have not resolved progenitors from NSCs.
Our specific aim has been to resolve NSC and progenitor populations in a clear and convincing manner using comprehensive immunostaining protocols with commercially available markers used in the field. Previously, we had identified NSCs at the onset of cortical development based on their complete lack of surface expression of several known neural markers (tetanus toxin (TxTx) fragment C and cholera toxin B complex ganglioside binding sites, A2B5 (GD3 ganglioside) and CD15/LeX/SSEA-1). We have added surface (CD133, NG2, PSA-NCAM, CD24, CD57, NGFR p75, integrin alpha 3, integrin alpha 6, integrin beta 1), cytoskeletal/cytoplasmic (beta 4 tublin, alpha 1 tubulin, Aldefluor) and nuclear markers (HuD, Ngn1, Ngn3, Mcm2, FoxG1, D1x2, Olig2, pax6, prox1, Sox1, Sox2, Hoechst), which are all of used in the field. We combined pairs of alpha and beta integrin subnits with CD57, a pan-specific marker of CD15+ CD24+ CDw60+ PSA-NCAM+ A2B5+ neural progenitors, and TnTx, a pan-specific neuronal lineage marker. This novel combination provided an unprecedented level of resolution and revealed the lineal relationships among all the major populations throughout corticogenesis. The co-expression of six surface markers effectively links six disparate literatures on neural progenitors together for the first time into the same lineage. Since these cells also co-expressed the transcription factor Tbr2, we identified them as intermediate progenitor cells (IPCs), thereby establishing multiple IPC-related surface markers. NSCs were identified as CD57- TnTx- cells co-expressing specific integrin heterodimers. These cells rapidly decreased in relative abundance during the early period of corticogenesis as they either self-renewed, died via apoptosis or differentiated into different CD57+/- and TnTx+ neural progenitors. We also included a live-cell DNA stain to quantify total DNA content. The most proliferative subsets were found in IPCs expressing heterodimers, while the most abundant post-mitotic cells were found in the neuronal progenitors devoid of either integrin subunit. This comprehensive surface phenotyping strategy provides, for the first time, a complete ex vivo account of the proliferating, quiescent/differentiating and apoptotic phases that accompany the intermediate stages of lineage progressions derived from NSCs. Analysis of the four bivariate FACS plots (CD57+/- TnTx+/-) associated with the four integrin subunit-labeled populations (alpha+/- beta+/-) revealed clear linkages among each of the neural populations either devoid of markers (CD57- TnTx- NSCs) or expressing either one or both markers. We conclude that NSCs initially give rise to both neuronal progenitors (CD57- TnTx+) and IPCs (CD57+ TnTx-), which , in turn, produce other neuronal progenitors (CD57+ TnTx+). The surface markers allowed us to sort select populations for retrospective analysis of their seminal properties using clonal cultures. The results obtained in vitro closely recapitulate the lineage progressions inferred from FACS analyses of populations phenotyped ex vivo, indicating that seminal properties are effectively preserved and hence accessible for cellular and molecular studies. NSCs self-renew and first produce IPC-lineage-negative neuronal progenitors followed by IPCs and their neuronal progenitor progeny. Subsequently, astrocytes, both lineage-negative and lineage-positive, are produced along with lineage-negative radial glial cells, which themselves give rise to lineage-negative neurons and astrocytes. It is clear that the seminal properties, including self-renewal, apoptosis, and multipotent differentiation, are all expressed by NSCs, IPCs and radial glia. Thus, each of these distinct, lineally-related phenotypes exhibits similar, but not identical seminal properties typically ascribed to NSCs. We also quantified the distributions of three other surface markers (CD133, NG2, NGFR p75) in the context of the beta integrin subunit, CD57 and TnTx. Each of these markers is expressed by rare populations throughout development (0-7%). CD133+ cells primarily reside at the ventricular interface in proliferating cells and may only be expressed at specific stage(s) of the cell cycle. The other two markers are mostly expressed by cells devoid of neural markers. Survey of several cytoplasmic markers revealed that alpha 1 tubulin is primarily expressed by IPCs and thus equates them with """"""""short neural precursors"""""""" in the literature, while beta 4 tubulin is restricted to oligodendroglial progeny. Neither Aldefluor labeling nor Hoechst dye exclusion were specific, since subpopulations of NSCs and IPCs were found to be Aldefluor+ and excluded Hoechst dye. Survey of NSC-associated transcription factors (TFs) showed that Pax6, Sox1 and Sox2 label the majority of NSCs and IPCs. These TFs are also expressed by radial glia, but are down-regulated among the differentiating neuronal phenotypes generated by these three proliferating populations. Other TFs (HuD, Ngn1, Ngn3, Prox1) emerge primarily among differentiating neuronal progenitors, especially those derived from IPCs, while Mcm2 and FoxG1 are co-expressed by these cells. In sum, lineage-negative NSCs self-renew and predominate at the earliest stages of corticogenesis and generate neuronal progenitors and IPCs, which differentiate into transiently-expressed pioneer neurons and transiently-expressed Cajal-Retzius neurons, respectively. Later IPCs generate the permanent pyramidal neurons composing the cortical layers. Self-renewing NSCs also differentiate into radial glia, which give rise to pyramidal neurons and astroglia. All of these cells co-express integrin subunits at varying levels. Our analysis has revealed 20 distinct populations with some transiently appearing, then disappearing and others persisting during cortical development. Using multi-marker phenotyping thus provides the first complete characterization of all emerging populations and reveals the dynamic lineal relationships that exist among them. This novel, all-inclusive framework is based on results using a tool kit of commercially available reagents, thus making the strategy accessible to all investigators interested in neural stem and progenitor cell biology.
| Su, Tung-Ping; Zhang, Lei; Chung, Ming-Yi et al. (2009) Levels of the potential biomarker p11 in peripheral blood cells distinguish patients with PTSD from those with other major psychiatric disorders. J Psychiatr Res 43:1078-85 |
| Nielsen, Joseph A; Lau, Pierre; Maric, Dragan et al. (2009) Integrating microRNA and mRNA expression profiles of neuronal progenitors to identify regulatory networks underlying the onset of cortical neurogenesis. BMC Neurosci 10:98 |
