Neurofibromatosis is caused by the loss of neurofibromin (Nf1), leading to peripheral nervous system (PNS) tumors, including neurofibromas and malignant peripheral nerve sheath tumors (MPNSTs). A long-standing question has been whether these tumors arise from neural crest stem cells (NCSCs) or differentiated glia. Germline or conditional Nf1 deficiency caused a transient increase in NCSC frequency and self-renewal in most regions of the fetal PNS. However, Nf1-deficient NCSCs did not persist postnatally in regions of the PNS that developed tumors and could not form tumors upon transplantation into adult nerves. Adult P0a-Cre+Nf1(fl/-) mice developed neurofibromas, and Nf1(+/-)Ink4a/Arf(-/-) and Nf1/p53(+/-) mice developed MPNSTs, but NCSCs did not persist postnatally in affected locations in these mice. Tumors appeared to arise from differentiated glia, not NCSCs.

Canonical Wnt signaling instructively promotes sensory neurogenesis in early neural crest stem cells (eNCSCs) (Lee, H.Y., M. Kléber, L. Hari, V. Brault, U. Suter, M.M. Taketo, R. Kemler, and L. Sommer. 2004. Science. 303:1020–1023). However, during normal development Wnt signaling induces a sensory fate only in a subpopulation of eNCSCs while other cells maintain their stem cell features, despite the presence of Wnt activity. Hence, factors counteracting Wnt signaling must exist. Here, we show that bone morphogenic protein (BMP) signaling antagonizes the sensory fate-inducing activity of Wnt/β-catenin. Intriguingly, Wnt and BMP act synergistically to suppress differentiation and to maintain NCSC marker expression and multipotency. Similar to NCSCs in vivo, NCSCs maintained in culture alter their responsiveness to instructive growth factors with time. Thus, stem cell development is regulated by combinatorial growth factor activities that interact with changing cell-intrinsic cues.

Schwann cells develop from multipotent neural crest stem cells and are important for neuronal survival, maintenance of axonal integrity, and myelination. We used transgenic mice expressing green fluorescent protein in a tissue-specific manner to isolate viable, pure populations of neural crest stem cells and developing Schwann cells, which are not readily accessible by microdissection. Starting with the minute amounts of RNA obtained, a two-round amplification procedure was used to achieve reproducible DNA array hybridizations. We validated our screening procedure by comparisons with the literature and by in situ hybridization. Stage-to-stage comparisons and hierarchical clustering for neural crest and five stages of Schwann cell development suggest a wealth of candidates for genes involved in stem cell regulation and in early Schwann cell development. The combination of methods applied in this study should be generally useful for isolating and profiling other stem cell and difficult to isolate cell populations.

The Polycomb gene Bmi-1 is required for the self-renewal of stem cells from diverse tissues, including the central nervous system (CNS). Bmi-1 expression is elevated in most human gliomas, irrespective of grade, raising the question of whether Bmi-1 over-expression is sufficient to promote self-renewal or tumorigenesis by CNS stem/progenitor cells. To test this we generated Nestin-Bmi-1-GFP transgenic mice. Analysis of two independent lines with expression in the fetal and adult CNS demonstrated that transgenic neural stem cells formed larger colonies, more self-renewing divisions, and more neurons in culture. However, in vivo, Bmi-1 over-expression had little effect on CNS stem cell frequency, subventricular zone proliferation, olfactory bulb neurogenesis, or neurogenesis/gliogenesis during development. Bmi-1 transgenic mice were born with enlarged lateral ventricles and a minority developed idiopathic hydrocephalus as adults, but none of the transgenic mice formed detectable CNS tumors, even when aged. The more pronounced effects of Bmi-1 over-expression in culture were largely attributable to the attenuated induction of p16Ink4a and p19Arf in culture, proteins that are generally not expressed by neural stem/progenitor cells in young mice in vivo. Bmi-1 over-expression therefore has more pronounced effects in culture and does not appear to be sufficient to induce tumorigenesis in vivo.

Dramatic progress has been made over recent years toward the elucidation of the mechanisms regulating lineage determination and cell survival in the developing peripheral nervous system. However, our understanding of Schwann cell development is limited. This is partly due to the difficulties in culturing primary Schwann cell precursor cells, the earliest developmental stage of the Schwann cell lineage defined to date. Both the inability to maintain cultured Schwann cell precursor cells in an undifferentiated state and the technical difficulties involved in their isolation have hampered progress. We have conditionally immortalized rat Schwann cell precursor cells using a retrovirally encoded EGFR/neu fusion protein to circumvent these problems and to generate a source of homogeneous cells. The resulting SpL201 cell line expresses p75 and nestin, two proteins expressed by neural crest-derived cells, as well as peripheral myelin protein 22, protein zero, and Oct-6 as markers of the Schwann cell lineage. When cultured in EGF-containing medium, the SpL201 cells proliferate and maintain an undifferentiated, Schwann cell precursor cell-like state. The cell line is dependent on EGF for survival but can differentiate into early Schwann cell-like cells in response to exogenous factors. Like primary rat Schwann cells, SpL201 cells upregulate Oct-6 and myelin gene expression in response to forskolin treatment. Furthermore, the SpL201 cell line can form myelin in the presence of axons in vitro and is capable of extensively remyelinating a CNS white matter lesion in vivo. Thus, this cell line provides a valuable and unique tool to study the Schwann cell lineage, including differentiation from the Schwann cell precursor cell stage through to myelination.

No abstract

SUMMARY Tumor-initiating cells have been suggested to be rare in many cancers. We tested this in mouse malignant peripheral nerve sheath tumors (MPNSTs) and found that 18% of primary and 49% of passaged MPNSTs cells from Nf1+/−;Ink4a/Arf−/− mice formed tumors upon transplantation whereas only 1.8% to 2.6% of MPNSTs cells from Nf1+/−;p53+/− mice did. MPNST cells of both genotypes require laminin binding to β1-integrin for clonogenic growth. Most MPNST cells from Nf1+/−;Ink4a/Arf−/− mice expressed laminin whereas most MPNST cells from Nf1+/−;p53+/− mice did not. Exogenous laminin increased the percentage of MPNST cells from Nf1+/−;p53+/−, but not Nf1+/−;Ink4a/Arf−/−, mice that formed tumorigenic colonies. Tumor-forming potential is therefore common among MPNST cells but the assay conditions required to detect it vary with tumor genotype.

No abstract