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PTH-046 Enteric nervous system stem cell-containing neurospheres: all that glitters is not gold?
  1. E Binder,
  2. M Cananzi,
  3. A J Burns,
  4. N Thapar
  1. Gastroenterology Unit, University College London Institute of Child Health and Great Ormond Street Hospital, London, UK

Abstract

Introduction Enteric nervous system (ENS) stem cells provide potential therapeutic tools for hitherto incurable gut neuropathies. Recent progress confirms they can be generated from human gut in cell aggregates or neurospheres (NS), but numerous challenges remain. Although previous studies have suggested that NS are a source of putative stem cells, can be successfully propagated and expanded in culture, and transplanted, few have assessed their purity and implications for clinical use. The aim of this study was to characterise the cellular content of NS generated and propagated in culture and identify putative cellular markers for better selective isolation or enrichment of ENS stem cells for therapy.

Methods Transgenic Wnt1-Cre/Floxed-YFP mice (express yellow fluorescent protein (YFP) in all Neural Crest (NC)-derived and ENS cells) were used. FACS-selection of YFP-positive cells from dissociated gut was used to isolate NC-derived cells to generate NS, which were then compared to those from YFP-negative (non-NC) or unsorted (mixed – akin to current protocols for human ENS stem cells) gut cells. Using immunostaining and clonogenic studies putative markers were then quantitatively assessed across all cultures for their ability to enrich for ENS stem cells, and studies applied to human tissue.

Result All cell fractions (unsorted (US), sorted YFP-positive (SoP) and sorted YFP-negative (SoN)) contained cells capable of generating NS. US NS showed partial YFP fluorescence unlike those from SoP (pure YFP) and SoN (no YFP). SoP NS appeared perfectly spherical unlike the others which were irregular and formed faster. NS characterisation showed that US NS contained both NC cells (YFP positive+any of-Tuj1, GFAP, p75, Sox10) and significant numbers of non-NC cells (positive for any of SMA, c-kit, cytokeratin). SoP NS contained NC cells only and SoN NS non-NC cells only. Both the precursor marker Nestin and proliferation marker PH3 were positive in both NC and non-NC cells although PH3 showed higher expression in non-NC cells. NS from all sources could be propagated and expanded in culture over multiple passages, but this did not alter the mixed (NC/non-NC) cellular content of US NS. The low affinity neurotrophin receptor p75 could be used to FACS-sort YFP-positive cells from dissociated gut (mixed NC/non-NC), and was also expressed in NS generated from human gut.

Conclusion Current protocols to generate ENS stem cell-containing NS for transplantation need refinement to reduce contamination with proliferating non-NC cells, which may carry safety implications if inadvertently transplanted in conditions of specific neuronal loss (eg, Hirschsprung's disease). Putative markers (eg, p75) may enable selective isolation or enrichment of ENS stem cells for clinical use.

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