262 Identification and Characterization of a Subset of Enteric “Glial” Cells As Neural Precursors

Abstract

Introduction: Recently, it has been shown that cells expressing the glial marker GFAP can make enteric neurons, raising the possibility that enteric glia can function as enteric neural precursor cells (ENPC). However, it is not known whether all or only a sub-population of enteric glia are ENPC. Apart from glial markers, ENPC are also known to express Nestin and p75NTR. Hence, using a Nestin-GFP mouse, we studied whether ENPC, defined by the co-expression of Nestin and p75NTR, express glial cell markers and whether all enteric glial cells express these ENPC markers. Methods: Cells from the longitudinal muscle-myenteric plexus (LMMP) of Nestin-GFP mice were flow sorted into four populations based on expression of Nestin-GFP and p75NTR for clonal proliferation and differentiation assays. Differentiated cells were tested for the presence of neurons and glia using immunocytochemistry. In separate analyses, these four cell populations were analyzed by FACS for the presence, co-localization, and intensity of fluorescence of glial markers GFAP and S100B. Results: By clonal analyses, we observe that only cells that co-express Nestin and p75NTRHI proliferate (Mean ± S.E. of percentage proliferative cells: 10.2 ± 0.62) and differentiate to form neurons and glia (Mean ± S.E. of percentage of neurospheres that differentiate to neurons and glia: 78.26 ± 4.35). ENPC, defined now by Nestin-p75NTRHI co-expression, express the glial markers, GFAP and S100B. We observe that only 3.86% of enteric glial cells that co-express GFAP and S100B express Nestin-GFP and p75HI and hence qualify for ENPC. All other cells are presumably mature enteric glia. Discussion: We show that ENPC co-express Nestin and p75NTRHI apart from GFAP and S100B, confirming earlier results that ENPC express glial markers. However, ENPC only form a small fraction of the total enteric glial cells as only 3.86% of cells that co-express the glial markers express Nestin-GFP and p75NTRHI. Hence, ENPC either represent a sub-category of enteric glial cells or are non-glial cells expressing shared markers. Our data shows that not all enteric glia are ENPC. This work paves the way for understanding not only the true identity of ENPC, but also that of mature enteric glia.