Fig 2: Immunofluorescent characterization of co-expression of OL pathway-specific markers.To further determine whether the O4-positive cells were co-expressing other OL-specific markers (OLIG2, SOX10, MBP, and O1) on the protein level, immunocytochemistry was performed for these protein markers, and co-expression was confirmed both in HC and SZ samples. Co-expression of OLIG2 (red) and O4 (green) in the same cells at differentiation D56 in HC (a) and SZ (b) samples. White arrows show examples of red OLIG2-positive nuclei in green O4-positive cells. SOX10 (red) and O4 (green) co-expression is shown at D65 in HC (c) and SZ (d). Myelin Basic protein (MBP) (green) and O1 (red) are seen at D85 in HC (e) and SZ (f). Note that yellow fluorescence indicates co-expression of the respective markers where both markers are predominantly in cytoplasm, as in panels c–f

Fig 3: Cell types present in neurosphere-like bodies. (A) Bar graph displaying the percentage of either SOX10+/p75+, HU+ or non–enteric nervous system (ENS) cells present in neurosphere-like bodies grown in nTCM with and without CHIR-99021 (no statistical difference was found between these groups). (B) A Z-stack optical section shows the characteristic staining of a SOX10+ and p75+, which were always found coexpressed in cells. Hu+ cells were often found in association with SOX10+/p75+ cells. However, the majority of cells did not stain for SOX10, p75, or Hu and were non-ENS population derived from the intestinal muscle.

Fig 4: Human enteric nervous system (ENS)–derived cells proliferate in vitro. (A) Incorporation of ethynyl-2'-deoxyuridine (EdU) (white arrowheads) into SOX10+ ENS cells with and without CHIR-99021. (B) Proliferation of SOX10+ cells is significantly increased by CHIR-99021, expressed as a ratio of EdU+/SOX+ cells to total SOX10+ cells. (C) Proliferation occurs in putative progenitor cells (EdU+/SOX10+/S100ß-ve; white arrowheads), but was not detected in neurons (HU+/SOX10-ve; open arrowhead) during the culture period. Images acquired using confocal microscopy. Data derived from two Hirschsprung disease (HSCR) patient samples.

Fig 5: rTSMS increases proliferation and modulates differentiation of spinal cord stem cells. (A) Experimental design, SCIs were performed on mice (day 0), then half of the mice has been treated using rTSMS during 2 weeks (from day 1 to day 14). At day 15, mice were fixed and neurosphere assays (B) or immunohistological analyses (C–K) were performed. (B) Quantification of the total number of neurospheres derived from 100,000 cells from control (SCI) and Stm (rTSMS treated) mice 15 days after injury. N = 4 animals per group. (C) Quantification of mean fluorescence intensity of tomato+ cells 15 days after SCI. (D, E, F, G, I, J) Representative pictures of axial spinal cord sections of (D, F, I) control and (E, G, J) Stm animals 15 days after SCI. Sections were stained with tomato and DAPI (D, E). Scale bars are 250 µm. (F–K) Identification of cells derived from Tomato+ recombined ependymal cells were identified by GFAP (F, G) and Sox10 (I, J). (H, K) Quantification of double positive recombined cells in the spinal cord 15 days after SCI. Scale bars are 100 µm. N = 5 animals per group. Quantifications are expressed as average ± SEM. * = p < 0.05