Volume 137, Issue 6, Pages 2063-2073.e4 (December 2009) Expansion and Differentiation of Neural Progenitors Derived From the Human Adult Enteric Nervous System  Marco Metzger, Petra M. Bareiss, Timm Danker, Silvia Wagner, Joerg Hennenlotter, Elke Guenther, Florian Obermayr, Arnulf Stenzl, Alfred Koenigsrainer, Thomas Skutella, Lothar Just  Gastroenterology  Volume 137, Issue 6, Pages 2063-2073.e4 (December 2009) DOI: 10.1053/j.gastro.2009.06.038 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 Enteric human spheroids isolated from adult gut and cultured for up to 2 weeks under proliferation conditions. (A) Bright-field view of enterospheres after 7 days in vitro derived from the rectum of a 66-year-old patient. (B) Higher magnification of a representative sphere. (C) Human enterospheres were incubated for 9 days with BrdU during the expansion phase. Whole-mount immunohistochemistry for BrdU (red). Nuclei were stained with Sytox green (green). (D) Higher magnification of a single sphere. (E) Immunocytochemistry for the intermediate filament nestin (red); nuclei (green). (F) p75 immunocytochemistry (red); nuclei (green). Yellow rectangles represent a magnified area (marked by the white rectangle) for the green (nuclei) and red (p75) fluorescence channel. Arrows indicate identical cells. Scale bars: A, 200 μm; B, C, E, and F, 50 μm; D, 25 μm. Gastroenterology 2009 137, 2063-2073.e4DOI: (10.1053/j.gastro.2009.06.038) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 (A) Distribution of cultured spheres after 6 and 9 days under proliferation culture conditions with respect to numbers and average diameter. A shift in sphere size can be observed between the 2 culture time points. (B) Quantification of BrdU-positive cells per sphere. Spheroid cultures were expanded for 14 days, and BrdU was added to the culture medium for the last 12 days. Box-and-whiskers graph represents data from 2 different tissue samples (sample 1, n = 10; sample 2, n = 25). The box extends from the 25th to the 75th percentiles, the middle line represents the median, and the whiskers extend to the lowest and highest data points. The asterisk indicates statistically significant differences between both groups analyzed by Mann–Whitney U test (P < .001). (C) Effect of conditioned medium on growth of human enterospheres. Preselected spheres with diameters between 100 and 150 μm were cultured for up to 11 days with culture medium supplemented with 50% (n = 8), 10% (n = 7), and 0% (n = 7) of conditioned supernatant of fetal cultures. Spheroid diameters and volumes were determined after 8 and 11 days in vitro. Values after 11 days in culture were normalized in percentage to sphere volumes after 8 days in vitro for each group. Data are expressed as means ± SD. The asterisks indicate statistically significant differences between day 8 and day 11 of each group analyzed by analysis of variance followed by Fisher's protected least significance difference test (P < .01). Gastroenterology 2009 137, 2063-2073.e4DOI: (10.1053/j.gastro.2009.06.038) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 Immunocytochemical characterization of differentiated human progenitor cells from adult gut. After expansion for 7–14 days, spheroids were differentiated for up to 4 weeks. (A) β-tubulin III (TuJ1) immunofluorescence of differentiated neurons (red); nuclei were stained with DAPI (blue). (B) p75 immunocytochemical staining (red); nuclei (blue). (C) S100β immunofluorescence of glial cells (green); nuclei (blue). (D) Positive stained cells for choline acetyl transferase (ChAT, red); nuclei (blue). (E) Serotonin (Ser) immunofluorescence of differentiated neurons (red); nuclei (blue). (F) Combined immunocytochemistry for smooth muscle actin (SMA, green) and TuJ1 (red); nuclei (blue). Scale bars: A–F, 50 μm. Gastroenterology 2009 137, 2063-2073.e4DOI: (10.1053/j.gastro.2009.06.038) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 Differentiated neural and smooth muscle cells were derived from proliferated BrdU-positive cells. At the end of the expansion phase, human enterospheres were incubated for 1 hour with BrdU and afterwards differentiated for 1 week. (A and B) Combined immunocytochemistry for β-tubulin III (TuJ1) (red) and BrdU (green); DAPI (blue). Arrows indicate the same neuronal cells. (C and D) Combined immunocytochemistry for glial fibrillary acid protein (GFAP, red) and BrdU (green); DAPI (blue). The arrows point to the same cell. (E and F) Combined immunocytochemistry for smooth muscle actin (SMA, red) and BrdU (green); DAPI (blue). The arrows mark the same cells. Scale bars: A–F, 25 μm. Gastroenterology 2009 137, 2063-2073.e4DOI: (10.1053/j.gastro.2009.06.038) Copyright © 2009 AGA Institute Terms and Conditions

Figure 5 Differentiated PGP9.5- and S100β-positive cells derived from proliferated BrdU-positive cells. Human adult enterospheres were incubated for 7 days with BrdU during the expansion phase and differentiated for 2 weeks. Immunocytochemistry for BrdU was performed with HCl pretreatment. (A) Combined immunocytochemistry for PGP9.5 (red) and BrdU (green). (B) Combined immunocytochemistry for S100β (red) and BrdU (green). Arrows indicate the double-stained cells, and arrowheads indicate cells that are only positive for BrdU. Scale bars: A, 10 μm; B, 25 μm. Gastroenterology 2009 137, 2063-2073.e4DOI: (10.1053/j.gastro.2009.06.038) Copyright © 2009 AGA Institute Terms and Conditions

Figure 6 (A) Qualitative RT-PCR analysis for p75 and RET of proliferating and differentiated enterospheres. Spheres were proliferated for 2 weeks and subsequently differentiated for 2 weeks. (B) Qualitative RT-PCR analysis for nitric oxide synthase 1 (NOS1) and tyrosine hydroxylase (TH) of differentiated enterospheres. (C) Quantitative real-time RT-PCR analysis of neurospheres proliferated for 2 weeks in comparison with expanded neurospheres 1 week after their differentiation. To reduce the proliferation of nonneuronal cells under differentiation conditions, fetal calf serum was omitted from the cell culture medium. Agarose gel electrophoresis of RT-PCR products: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), microtubule-associated protein 2 (MAP2), and neurofilament medium protein (NF). Complementary DNA of human gut served as positive control (pos. c.). RNA of cultures was used as negative control (neg. c.). Gastroenterology 2009 137, 2063-2073.e4DOI: (10.1053/j.gastro.2009.06.038) Copyright © 2009 AGA Institute Terms and Conditions

Figure 7 Electrophysiological analysis of differentiated enteric neurons. (A) Bright-field view of a patch-clamped neuron (arrow). The arrowhead points to the electrode. The analyzed cell was positively immunostained for β-tubulin III (TuJ1; B, green) and nitric oxide synthase (NOS; C, red); DAPI (blue). (D and E) Spheroid-derived neuron exhibited functional voltage-dependent sodium channels. (D) Family of whole cell sodium currents. (E) Current-voltage [I-V] relationships of the peak inward currents shown in D. Scale bars: A–C, 25 μm. Gastroenterology 2009 137, 2063-2073.e4DOI: (10.1053/j.gastro.2009.06.038) Copyright © 2009 AGA Institute Terms and Conditions

Figure 8 Implantation of enterospheres into intestinal organotypic slice cultures of fetal mice. (A) Bright-field view of an intestinal slice culture on day 1. The arrow points to the center of the culture where the enteric spheres were implanted. The dashed line indicates the border of the muscle tissue. (B–F) Combined immunofluorescence against p75 (green) and in situ hybridization for human-specific Alu sequences (red). (B) Bright-field and (C and D) fluorescence views of a slice culture on day 8. The asterisk shows the implantation site. The broken line denotes the border of the smooth muscle cells, and the solid line denotes the border of the culture. The arrows point to migrated cells. (D) Higher magnification of C. (E) A higher magnification of migrated cells with neural-like extensions. (F) Paraffin cross section of implanted intestinal slice culture, combined bright-field/fluorescence view. m, the muscle cell area of tissue culture. Scale bars: A–C, 100 μm; D and E, 50 μm; F, 20 μm. Gastroenterology 2009 137, 2063-2073.e4DOI: (10.1053/j.gastro.2009.06.038) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 1 Immunohistological evaluation of antibodies used in this study on cryostat cross sections from neonatal human colon. The microscopic views demonstrate the myenteric plexus located between the circular and longitudinal muscle layers. (A and B) Immunohistochemistry with the neuronal markers β-tubulin III (TuJ1) (red; A) and PGP9.5 (red; B); nuclei were stained with DAPI (blue). (C and D) Immunohistochemical stainings against the glial antigens glial fibrillary acid protein (GFAP) (red; C) and S100β (red; D); nuclei (blue). (E) Neurotrophin receptor p75 staining (red); nuclei (blue). (F) Nestin immunofluorescence (red). (G) Smooth muscle actin (SMA) immunohistochemistry (red; G); nuclei (blue). (H and I) Immunoglobulin G–negative controls for (H) mouse and (I) rabbit; nuclei (blue). Scale bars: A–I, 50 μm. Gastroenterology 2009 137, 2063-2073.e4DOI: (10.1053/j.gastro.2009.06.038) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 2 Whole-mount immunofluorescence of proliferating adult human enterospheres with immunoglobulin G–negative controls. (A) Immunoglobulin G mouse control (red). Nuclei were stained with Sytox green (green). (B) Immunoglobulin G rabbit control (red); nuclei (green). Scale bars: A and B, 50 μm. Gastroenterology 2009 137, 2063-2073.e4DOI: (10.1053/j.gastro.2009.06.038) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 3 Spheroid cell sprouting was reduced using differentiation medium without fetal calf serum. Adult human spheres were cultured for 2 weeks under proliferation conditions and subsequently differentiated for 2 weeks using differentiation cell culture medium. (A–C) Differentiation medium was supplemented with 2% fetal calf serum. A–C represent the same area. (D–F) Fetal calf serum was omitted from the differentiation medium. D–F represent the same area. (A and D) Bright-field view. (B and E) Nucleus staining with DAPI (blue). (C and F) S100β immunocytochemistry (green); nuclei (blue). Scale bars: A–F, 40 μm. Gastroenterology 2009 137, 2063-2073.e4DOI: (10.1053/j.gastro.2009.06.038) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 4 Implantation of human enterospheres into aganglionic explants from embryonic distal colon (E11.0). (A) Bright-field view of an explant with an integrated enterosphere (arrows) 2 days after implantation. (B) Higher magnification of A. (C) Combined whole-mount immunofluorescence for p75 (green) and human nuclei (red) of the implanted enterosphere (arrows) shown in A 3 days after grafting. (D) Higher magnification of C. The arrow points to a neuronal-like neurite of a p75/human nuclei costained cell. The arrowhead marks a p75-negative human cell. (E) Combined immunocytochemistry against p75 (green) and human nuclei (red) of an implanted enterosphere 6 days after grafting. Nuclei were stained with TOPRO (blue). Scale bars: A, 100 μm; B, 50 μm; C, 25 μm; D, 10 μm; E, 20 μm. Gastroenterology 2009 137, 2063-2073.e4DOI: (10.1053/j.gastro.2009.06.038) Copyright © 2009 AGA Institute Terms and Conditions