Jack T. Mosher

Neural Crest Stem Cells Persist in the Adult Gut but Undergo Changes in Self-Renewal, Neuronal Subtype Potential, and Factor Responsiveness

We found neural crest stem cells (NCSCs) in the adult gut. Postnatal gut NCSCs were isolated by flow-cytometry and compared to fetal gut NCSCs. They self-renewed extensively in culture but less than fetal gut NCSCs. Postnatal gut NCSCs made neurons that expressed a variety of neurotransmitters but lost the ability to make certain subtypes of neurons that are generated during fetal development. Postnatal gut NCSCs also differed in their responsiveness to lineage determination factors, affecting cell fate determination in vivo and possibly explaining their reduced neuronal subtype potential. These perinatal changes in gut NCSCs parallel perinatal changes in hematopoietic stem cells, suggesting that stem cells in different tissues undergo similar developmental transitions. The persistence of NCSCs in the adult PNS opens up new possibilities for regeneration after injury or disease.

Cell-Intrinsic Differences between Stem Cells from Different Regions of the Peripheral Nervous System Regulate the Generation of Neural Diversity

Stem cells in different regions of the nervous system give rise to different types of mature cells. This diversity is assumed to arise in response to local environmental differences, but the contribution of cell-intrinsic differences between stem cells has been unclear. At embryonic day (E)14, neural crest stem cells (NCSCs) undergo primarily neurogenesis in the gut but gliogenesis in nerves. Yet gliogenic and neurogenic factors are expressed in both locations. NCSCs isolated by flow-cytometry from E14 sciatic nerve and gut exhibited heritable, cell-intrinsic differences in their responsiveness to lineage determination factors. Gut NCSCs were more responsive to neurogenic factors, while sciatic nerve NCSCs were more responsive to gliogenic factors. Upon transplantation of uncultured NCSCs into developing peripheral nerves in vivo, sciatic nerve NCSCs gave rise only to glia, while gut NCSCs gave rise primarily to neurons. Thus, cell fate in the nerve was stem cell determined.

Neural crest stem cells undergo multilineage differentiation in developing peripheral nerves to generate endoneurial fibroblasts in addition to Schwann cells

Neural crest stem cells (NCSCs) persist in peripheral nerves throughout late gestation but their function is unknown. Current models of nerve development only consider the generation of Schwann cells from neural crest, but the presence of NCSCs raises the possibility of multilineage differentiation. We performed Cre-recombinase fate mapping to determine which nerve cells are neural crest derived. Endoneurial fibroblasts, in addition to myelinating and non-myelinating Schwann cells, were neural crest derived, whereas perineurial cells, pericytes and endothelial cells were not. This identified endoneurial fibroblasts as a novel neural crest derivative, and demonstrated that trunk neural crest does give rise to fibroblasts in vivo, consistent with previous studies of trunk NCSCs in culture. The multilineage differentiation of NCSCs into glial and non-glial derivatives in the developing nerve appears to be regulated by neuregulin, notch ligands, and bone morphogenic proteins, as these factors are expressed in the developing nerve, and cause nerve NCSCs to generate Schwann cells and fibroblasts, but not neurons, in culture. Nerve development is thus more complex than was previously thought, involving NCSC self-renewal, lineage commitment and multilineage differentiation.

The Loss of Nf1 Transiently Promotes Self-Renewal but Not Tumorigenesis by Neural Crest Stem Cells

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.

Temporally Distinct Requirements for Endothelin Receptor B in the Generation and Migration of Gut Neural Crest Stem Cells

Loss of Endothelin-3/Endothelin receptor B (EDNRB) signaling leads to aganglionosis of the distal gut (Hirschsprungs disease), but it is unclear whether it is required primarily for neural crest progenitor maintenance or migration. Ednrb-deficient gut neural crest stem cells (NCSCs) were reduced to 40% of wild-type levels by embryonic day 12.5 (E12.5), but no further depletion of NCSCs was subsequently observed. Undifferentiated NCSCs persisted in the proximal guts of Ednrb-deficient rats throughout fetal and postnatal development but exhibited migration defects after E12.5 that prevented distal gut colonization. EDNRB signaling may be required to modulate the response of neural crest progenitors to migratory cues, such as glial cell line-derived neurotrophic factor (GDNF). This migratory defect could be bypassed by transplanting wild-type NCSCs directly into the aganglionic region of the Ednrb(sl/sl) gut, where they engrafted and formed neurons as efficiently as in the wild-type gut.

Genetic background impacts developmental potential of enteric neural crest-derived progenitors in the Sox10Dom model of Hirschsprung disease

Abnormalities in the development of enteric neural crest-derived progenitors (ENPs) that generate the enteric nervous system (ENS) can lead to aganglionosis in a variable portion of the distal gastrointestinal tract. Cumulative evidence suggests that variation of aganglionosis is due to gene interactions that modulate the ability of ENPs to populate the intestine; however, the developmental processes underlying this effect are unknown. We hypothesized that differences in enteric ganglion deficits could be attributable to the effects of genetic background on early developmental processes, including migration, proliferation, or lineage divergence. Developmental processes were investigated in congenic Sox10(Dom) mice, an established Hirschsprung disease (HSCR) model, on distinct inbred backgrounds, C57BL/6J (B6) and C3HeB/FeJ (C3Fe). Immuno-staining on whole-mount fetal gut tissue and dissociated cell suspensions was used to assess migration and proliferation. Flow cytometry utilizing the cell surface markers p75 and HNK-1 was used to isolate live ENPs for analysis of developmental potential. Frequency of ENPs was reduced in Sox10(Dom) embryos relative to wild-type embryos, but was unaffected by genetic background. Both migration and developmental potential of ENPs in Sox10(Dom) embryos were altered by inbred strain background with the most highly significant differences seen for developmental potential between strains and genotypes. In vivo imaging of fetal ENPs and postnatal ganglia demonstrates that altered lineage divergence impacts ganglia in the proximal intestine. Our analysis demonstrates that genetic background alters early ENS development and suggests that abnormalities in lineage diversification can shift the proportions of ENP populations and thus may contribute to ENS deficiencies in vivo.

LGR5 is Expressed by Ewing Sarcoma and Potentiates Wnt/β-Catenin Signaling.

Ewing sarcoma (ES) is an aggressive bone and soft tissue tumor of putative stem cell origin that predominantly occurs in children and young adults. Although most patients with localized ES can be cured with intensive therapy, the clinical course is variable and up to one third of patients relapse following initial remission. Unfortunately, little is yet known about the biologic features that distinguish low-risk from high-risk disease or the mechanisms of ES disease progression. Recent reports have suggested that putative cancer stem cells exist in ES and may contribute to an aggressive phenotype. The cell surface receptor leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) is a somatic stem cell marker that functions as an oncogene in several human cancers, most notably colorectal carcinoma. LGR5 is a receptor for the R-spondin (RSPO) family of ligands and RSPO-mediated activation of LGR5 potentiates Wnt/β-catenin signaling, contributing to stem cell proliferation and self-renewal. Given its presumed stem cell origin, we investigated whether LGR5 contributes to ES pathogenesis. We found that LGR5 is expressed by ES and that its expression is relatively increased in cells and tumors that display a more aggressive phenotype. In particular, LGR5 expression was increased in putative cancer stem cells. We also found that neural crest-derived stem cells express LGR5, raising the possibility that expression of LGR5 may be a feature of ES cells of origin. LGR5-high ES cells showed nuclear localization of β-catenin and robust activation of TCF reporter activity when exposed to Wnt ligand and this was potentiated by RSPO. However, modulation of LGR5 or exposure to RSPO had no impact on proliferation confirming that Wnt/β-catenin signaling in ES cells does not recapitulate signaling in epithelial cells. Together these studies show that the RSPO-LGR5-Wnt-β-catenin axis is present and active in ES and may contribute to tumor pathogenesis.

Crossing the boundaries of sensory neurogenesis

Boundary cap cells of the dorsal root ganglia were thought to be limited to a structural role regulating migration into or out of the neural tube. Now a study in this issue reports that they are progenitors of small-diameter nociceptive neurons.

Abstract 5033: Modeling Ewing's sarcoma and tolerance of EWS-FLI1 with neural crest stem cells.

Ewing9s sarcoma (ES) is a highly malignant bone and soft tissue tumor that primarily affects children and young adults. These tumors are characterized by chromosomal translocations, most commonly t(11;22)(q24;q12), which generates the EWS-FLI1 fusion oncogene. Despite its oncogenic potential, the fusion protein induces cell cycle arrest or apoptosis in most non-transformed cells unless mutations in tumor suppressors such as p16 are also present. However, only a minority of patients (∼25%) have such mutations at diagnosis, suggesting other mechanisms for cellular tolerance must be present in the cell of origin. Currently, the cell of origin of these tumors is controversial, with data supporting both a mesenchymal and/or neural crest origin. To assess the potential to model ES using mouse neural crest stem cells (NCSCs) and elucidate the mechanism(s) of EWS-FLI1 tolerance, primary fetal and adult mouse NCSCs were transduced with a V5-tagged EWS-FLI1 2a eGFP construct. Both wild-type fetal and postnatal NCSCs tolerated EWS-FLI1 expression, with 40-60% of the stem cell-derived neurospheres positive for GFP. There was no difference in neurosphere forming ability between cells infected with EWS-FLI1 or an empty vector, suggesting EWS-FLI1 expression was not toxic to these cells. EWS-FLI1 expression was maintained as the stem cells self-renewed, with GFP-positive primary neurospheres giving rise to GFP-positive secondary and tertiary neurospheres but there was no difference in the frequency of GFP-positive and -negative secondary neurospheres. EWS-FLI1 expression was confirmed by quantitative PCR and the V5 tag was localized to the nucleus by immunocytochemistry, consistent with transcription factor function. EWS-FLI1-mediated gene expression analysis is currently ongoing. Interestingly, the tolerance of EWS-FLI1 was dramatically reduced in Bmi-1 deficient NCSCs. Bmi-1 is known to function cooperatively with multiple other oncogenes to mediate transformation but its role in tolerance has not been studied. In the absence of Bmi-1 only 8% of the fetal neurospheres were positive for GFP, a large decrease compared to 40% of the wild-type neurospheres. Taken together, these data are consistent with a neural crest origin for ES and demonstrate a critical role for Bmi-1-mediated tolerance to EWS-FLI1. Work is ongoing to determine whether EWS-FLI1 can transform these cells and to assess the full utility of this novel system to model ES and genetically assess important regulators of EWS-FLI1 function. Citation Format: Jack T. Mosher, Victor S. Chen, Elizabeth R. Lawlor. Modeling Ewing9s sarcoma and tolerance of EWS-FLI1 with neural crest stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5033. doi:10.1158/1538-7445.AM2013-5033