Enteric Neurospheres Are Not Specific to Neural Crest Cultures: Implications for Neural Stem Cell Therapies

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Neurospheres can be generated from YFP+ve, YFP-ve and mixed gut cell populations.

<p>Panels show phase contrast images of cultures containing neurospheres and their YFP fluorescent counterparts. (A-D) Neurospheres from unsorted gut cells contain both YFP+ve and YFP-ve cells whereas neurospheres derived from YFP+ve sorted cells (E-H) contain only YFP+ve cells. (I-L) Neurospheres from cultures of YFP-ve, non-neural crest cells do not contain YFP+ve cells. Neurospheres are similar in size and appearance in all cases. Scale bar in G = 200μm (applies to A-C, E-G, I-K) and in H = 40μm (applies to D, H, L).</p

Distribution, selection and proportion of YFP+ve cells in developing mouse gut.

<p>(A-D) Transverse sections of <i>Wnt1-Cre;Rosa26</i>^{<i>Yfp/Yfp</i>} mouse gut showing distribution of YFP+ve cells at the level of the presumptive myenteric (A-D) and submucosal (C,D arrowheads) plexuses. (E) FACS selection of YFP+ve cells. Bar graph shows the proportion of YFP+ve cells at selected stages of development. Scale bars = 50μm in A,B and 100μm in C,D.</p

Immunocytochemical characterization of neurospheres generated from sorted YFP+ve neural crest-derived cells.

<p>YFP+ve neurospheres contain cells positive for neural crest and neural stem/progenitor markers p75 (A), Sox10 (B) and Nestin (C), the neuronal marker TuJ1 (D), glial markers GFAP (E) and S100 (F), and the proliferation markers PH3 (G) and Ki67 (H, arrows). YFP+ve neurospheres do not contain cells expressing the ICC marker c-Kit (I) or the smooth muscle marker, SMA (J). All neurospheres show YFP+ve cells in green, corresponding markers in red and DAPI (nuclei) in blue. Scale bar = 50μm.</p

Transplantation of neurospheres into mouse gut.

<p>(A) Confocal images of host gut transplanted with YFP+ve neurospheres. YFP+ve cells spread from the site of transplantation and integrated with the endogenous ENS as shown by TuJ1 immunolabelling (red). (B) Host gut transplanted with YFP-ve neurospheres. YFP-ve cells, labelled with lentiviral construct expressing GFP (green), did not integrate with the endogenous ENS (red) and were apparent as individual, isolated cells. Scale bar = 50μm.</p

Neurospheres derived from postnatal human gut cultures are heterogeneous in composition.

<p>(A) Bright field image of neurosphere derived from human postnatal gut cultured for 2 weeks. Neurospheres contain neural crest-derived cells as shown by expression of the neural crest cell marker p75 (B) and the neuronal marker TuJ1 (C). Neurospheres also contained non-neural crest cell derivatives such as fibroblast-like cells expressing PDGFRα (D), and muscle cells expressing SMA (E). Proliferating cells are labelled with Ki67 (F). Following FACS sorting based on selection with p75^NTR, neurospheres were generated exclusively from p75-ve (G) and p75+ve cells (H) similar in shape and appearance. DAPI (blue) stains nuclei in B-F. Scale bar = 50μm.</p

Characterization of neurospheres generated from YFP-ve, non-neural crest-derived cells.

<p>Neurospheres derived from YFP-ve cells do not contain cells expressing neural crest cell markers p75 (A), Sox10 (B), neuronal marker TuJ1 (D) or glial markers GFAP (E) or S100 (F). However they do contain cells expressing Nestin (C), proliferation markers PH3 (G, arrows), Ki67 (H, arrows), ICC marker c-Kit (I) and smooth muscle marker SMA (J). All markers in red and DAPI (nuclei) in blue. Scale bar = 50μm.</p