In Vitro Formation of Enteric Neural Network Structure in a Gut-Like Organ Diffe - 《stemcells》干细胞学杂志干细胞之家



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作者：Miyako Takakia, Shinsuke Nakayamab, Hiromi Misawaa, Tadashi Nakagawaa, Hiroki Kuniyasuc作者单位：a Department of Physiology II, Nara Medical University, School of Medicine, Kashihara, Nara, Japan;b Department of Physiology I, Nagoya University Graduate School of Medicine, Tsurumai, Nagoya, Japan;c Department of Molecular Pathology, Nara Medical University, School of Medicine, Kashihara, Nara, J

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      【摘要】% K, ^8 @+ _& W- g5 }
   Using an embryoid body (EB) culture system, we developed a functional organ-like cluster¡ªa "gut"¡ªfrom mouse embryonic stem (ES) cells (ES gut). Each ES gut exhibited spontaneous contractions but did not exhibit distinct peristalsis-like movements. In these spontaneously contracting ES guts, dense distributions of interstitial cells of Cajal (c-kit i) at single or multiple sites that were attenuated by atropine or abolished by tetrodotoxin. These results suggest in vitro formation of physiologically functioning enteric cholinergic excitatory neurons. We for the first time succeeded in the differentiation of functional neurons in ENS by exogenously adding BDNF in the ES gut, resulting in generation of distinct peristalsis-like movements. , O, K, `0 y8 A: G! w
      【关键词】 Brain-derived neurotrophic factor c-kit Proto-oncogene tyrosine-protein kinase receptor ret precursor Interstitial cells of Cajal Neurofilament Protein gene product Tyrosine kinase B receptor/ p8 i0 O7 a8 h7 M$ |
   INTRODUCTION" S& ^4 \( k; Z9 G7 K

Recently, embryonic stem (ES) cells were shown to spontaneously give rise to a functional organ-like unit, the "ES gut," which undergoes rhythmic contractions and is topographically comprised of enteric derivatives of all three embryonic germ layers: epithelial cells (endoderm), smooth muscle cells and interstitial cells of Cajal (ICCs; c-kit  cells) (mesoderm), and a small number of diffusely distributed enteric neurons (ectoderm) ., C1 N1 C0 g. F. t( M! p( C2 A: p
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Enteric neurons are also present within the GI tract; enteric neurons innervate the smooth muscle and are essential for peristalsis .
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The ENS is an independent nervous system that structurally resembles the "first" brain i) elicited by focal stimulation of enteric nervous circuits. Improved technology presented here could facilitate a significant advance in the formation of a complete gut-like organ for regenerative medicine.; p3 T1 `5 z8 E

MATERIALS AND METHODS

ES Cell Culture
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Undifferentiated ES cells (EB3) were maintained on gelatin-coated dishes without feeder cells in Dulbecco¡¯s modified Eagle¡¯s medium (Sigma, St. Louis, http://www.sigmaaldrich.com) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, http://www.invitrogen.com), 0.1 mM 2-mercaptoethanol (Wako Chemical, Osaka, Japan, http://www.wako-chem.co.jp/english), 0.1 mM nonessential amino acids (Gibco), 1 mM sodium pyruvate (BioWhittaker Molecular Applications, Rockland, ME, http://www.bmaproducts.com), and 1,000 U/ml of LIF (Chemicon, Temecula, CA, http://www.chemicon.com) .
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Analysis of Gut Motility

We monitored and recorded video images of ES guts using a microscope-video recording system (Olympus IX70 and Victor cassette recorder BR-S605B, Tokyo, http://www.olympus-global.com). In the videotaped images, we counted the number of spontaneous contractions per 5-minute period over the course of at least three 5-minute periods, while maintaining the temperature of the dish at 35¡ãC using a micro-warm plate system (U HP-100; Kitazato Supply, Tokyo, http://www.kitazato-supply.com) .
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Immunohistochemistry) N( ?0 r6 Q1 q  h
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For immunohistochemical detection of c-kit, a transmembrane receptor that has tyrosine kinase activity, whole-mount preparations of ES gut were fixed in acetone (4¡ãC, 5 minutes) .
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For immunohistochemistry of thin sections, ES gut was fixed with 4% paraformaldehyde at 4¡ãC and then scraped from the culture dish and embedded into 0.6% agarose-PBS, which was subsequently embedded in a paraffin block in the usual manner. Consecutive 4-µm sections were cut from each block, subjected to antigen retrieval with pepsin (DakoCytomation, Inc., Carpinteria, CA, http://www.dakousa.com) for 20 minutes at room temperature, and immunostained using the immunoperoxidase technique. After blockade of endogeneous peroxidase activity by incubation for 15 minutes in 3% H2O2-methanol, the sections were rinsed with PBS and incubated with a primary antibody diluted with Washing Solution (BioGenex, San Ramon, CA, http://www.biogenex.com) for 2 hours at room temperature. Thereafter, they were rinsed again with PBS and incubated for 1 hour at room temperature with a peroxidase-conjugated secondary antibody diluted to 0.5 µg/ml (Medical & Biotechnological Laboratories Co., Ltd., Nagoya, Japan, http:// http://www.mbl.co.jp). All sections were then rinsed with PBS, color-developed using diaminobenzidine (DAB) solution (DakoCytomation, Inc.), washed in water, and counterstained with Meyer¡¯s hematoxylin (Sigma). Care was taken to ensure that the antibody reaction and DAB exposure were the same for all specimens. The antibodies and working concentrations used in the primary reaction were anti-c-ret (proto-oncogene tyrosine-protein kinase receptor ret precursor) (N-term, 1 µg/ml; Abgent, San Diego, http://www.abgent.com), anti-mouse c-kit (2 µg/ml; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, http://www.scbt.com), anti-trkB (tyrosine kinase B receptor) (clone H-181, 1 µg/ml; Santa Cruz Biotechnology, Inc.), anti-NF (clone 2F11, reacting with 70, 160 and 200 kDa proteins, 0.5 µg/ml; DakoCytomation, Inc.), anti-sox9 (clone H-90, 0.5 µg/ml; Santa Cruz Biotechnology, Inc.), and anti-p75 (intracellular domain, 0.5 µg/ml; Upstate).

Ca2  Imaging

ES guts were incubated for 3¨C4 hours at room temperature in modified Krebs solution containing 10 µM fluo-3 acetoxy-methyl ester (Dojindo, Kumamoto, Japan, http://www.dojindo.com) and detergents (0.02% Pluronic F-127 i induced by focal stimulation (5¨C10 Hz, 100 µseconds, 10¨C20 pulses, variable voltage) applied through the Ag-wire inserted in a fine glass electrode; an Ag-plate served as the indifferent electrode.* J4 w! W. m1 l- @6 D9 z6 K
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Among the drugs used, tetrodotoxin (TTX), atropine, and hexamethonium were purchased from Sigma. All chemicals were dissolved in distilled water as stock solutions and further diluted as needed with Tyrode or Krebs solution to the desired concentrations. (The ratios of the dilution were always greater than 1:1,000.)

RESULTS& @$ t; n+ W/ Y) n; g  N
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ES Cell Culture& }/ v( ]: M# ]8 g, a$ {
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To induce differentiation, we placed ES cells in hanging-drop cultures with medium containing BDNF (10¨C9 to ~5 x 10¨C8g/ml)  on 100-mm gelatin-coated dishes and allowed to attach in the absence of BDNF. Apparently, addition of BDNF to the hanging-drop cultures was the key step leading to differentiation of ES guts containing enteric neural ganglia, because additional BDNF applied to outgrowth cultures had no effect on formation of enteric neural ganglia. We confirmed that NT-3 and GDNF at 10¨C9 to approximately 10¨C8 g/ml were also able to differentiate enteric nerve fibers and a few or a moderate number of cell bodies, but we did not confirm the differentiation of a single or multiple enteric neural ganglia (Fig. 1).- f4 ^; L2 p7 e% j: Y9 M, @# T
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Figure 1. Neurofilament immunoreactivity in embryonic stem cell guts differentiated from the embryoid body (GDNF  or NT-3 ) reveals a small number of nerve cells (A) and a network-like structure (B) but not any enteric neural ganglia. Abbreviations: GDNF, glial cell line-derived neurotrophic factor; NT-3, neurotrophin-3.

Migration of neural crest stem cells to the gut is dependent on transient expression of ETB receptors . However, differentiation of enteric neural ganglia, identified by NF and PGP 9.5 immunoreactivity, did not occur when an ETB receptor agonist, IRL-1620 (10¨C9 to ~2 x 10¨C9 M), was added to the hanging-drop cultures, the outgrowth cultures, or both.0 P3 M! O- R, `6 M
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Immunohistochemistry5 h0 p+ Y. U; g* y; a. Y

The timing and BDNF-dependence of the expression of tyrosine kinase B receptors encoded by the trk proto-oncogenes (trkB; BDNF receptors), proto-oncogene tyrosine-protein kinase receptor ret precursor (c-ret) . By contrast, we detected sox9 and p75 immunoreactivity only after 2 and 4 days in the hanging-drop culture, but we detected none of c-kit, NF, sox9, and p75 immunoreactivity in the EBs (after 6¨C7 days in the hanging-drop culture) (Fig. 2A, 2C; Table 1). In addition, no expression of trkB, c-ret, NF or PGP 9.5, sox9, and p75 was detected throughout the hanging-drop culture in the absence of BDNF (Fig. 2B, 2D; Table 1). Regardless of BDNF, after 6- to 7-day hanging-drop culture, the formation into the EB dramatically occurred (Fig. 2C, 2D). Simultaneously, migrating neural crest cell markers sox9 and p75 disappeared in the EB formed in the presence of BDNF (Fig. 2C; Table 1).

Table 1. Immunohistochemistry of trkB, c-ret, NF, c-kit, sox9, and p75# a2 e2 z( m9 [; ~/ v: a  r: ^
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Figure 2. Immunohistochemical detection of trkB, c-ret, NF, c-kit, sox9, and p75 after 4 days and 6¨C7 days (an EB) in hanging-drop culture and after 2¨C3 weeks in outgrowth culture. (A): Immunoreactivity associated with trkB, c-ret, sox9, and p75, but no NF or c-kit immunoreactivity, was identified after 4 days, treated with BDNF (BDNF ). (B): No trkB, c-ret, NF, c-kit, sox9, or p75 immunoreactivity was seen after 4 days, not treated with BDNF (BDNF¨C). (C): Immunoreactivity associated with trkB and c-ret, but no NF, c-kit, sox9, or p75 immunoreactivity, was identified in an EB treated with BDNF (BDNF ). (D): No trkB, c-ret, NF c-kit, sox9, or p75 immunoreactivity was seen in another EB not treated with BDNF (BDNF¨C). (E): c-ret immunoreactivity was apparent in an ES gut differentiated from EB (BDNF ) after 2 weeks in outgrowth culture but was absent after 3 weeks (top panels). No c-ret immunoreactivity was detected in ES guts differentiated from EB (BDNF¨C) after 2¨C3 weeks (bottom panels) in outgrowth culture. (F): trkB, c-ret, NF, and c-kit immunoreactivity in a BDNF ES gut after 2 weeks in outgrowth culture. The specimen shown is the same as in the left top panel of (E). Abbreviations: BDNF, brain-derived neurotrophic factor; EB, embryoid body; ES, embryonic stem; NF, neurofilament.7 W/ ^5 C( }  a) ]# f  ~3 k6 N

We also detected c-ret immunoreactivity in ES guts differentiated from EBs treated with BDNF (BDNF ) after 2 weeks in outgrowth culture but not after 3 weeks (Fig. 2E; Table 1). On the other hand, trkB, NF, and c-kit immunoreactivity was detected after 2 and 3 weeks in outgrowth culture (Fig. 2F). Within ES guts, NF immunoreactive ( ) cells formed a ganglion within the wall of the dome-like structure surrounding the lumen (Fig. 2F). No trkB, c-ret (Fig. 2E), or NF immunoreactivity was detected at any time in ES guts differentiated from EBs formed in the absence of BDNF (BDNF¨C), whereas c-kit immunoreactivity was identified regardless of BDNF treatment (Table 1).4 G  K: p3 N# S
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In whole-mount preparations of ES guts differentiated from BDNF  EBs, a group of NF  cells formed several ganglia within the walls of the dome-like structure that surrounds the lumen (Fig. 3A, 3B). Moreover, these ganglia connected by fiber tracts form an enteric neural network. Also, c-kit  cells were present. These mainly multipolar cells did not form a distinct layer but were instead scattered throughout the muscle layer (Fig. 3C, 3D), where they formed a dense network. As previously reported . Taken together, the data presented here suggest that BDNF specifically differentiates enteric neurons within ES guts from enteric neural crest-derived cells in the EB during the 2¨C3 weeks of outgrowth culture.1 ~) t0 n7 @; j) c' t# K' p
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Figure 3. NF (A, B) and c-kit (C, D) immunoreactivity in different contracting ES guts on day 21 of EB (BDNF ) culture. Arrows in (A) indicate enteric neural ganglia. (E): Series of video images showing a highly coordinated peristaltic contraction of an ES gut on day 21 of EB (BDNF ) culture. Abbreviations: BDNF, brain-derived neurotrophic factor; EB, embryoid body; ES, embryonic stem; NF, neurofilament.$ c% I1 R( D4 s% k
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Analysis of ES Gut Motility; [  e" v: h, x* Y1 f! m; l

By recording serial video images at various stages of differentiation, we were able to determine that ES guts typically begin to exhibit spontaneous contractions on day 21 of EB outgrowth culture. As shown in Figure 3E, a tubular (cystic structure) ES gut differentiated from a BDNF  EB exhibited distinct patterns of highly coordinated peristalsis-like contractions that differed from the phasic contractions seen in ES guts that spontaneously differentiated from a BDNF¨C EB. These peristalsis-like contractions occurred significantly less frequently than the rhythmic, often local, contractions that were also seen (Table 2).

Table 2. Differences in motility pattern of ES guts/ A% P' r5 M& g
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The Response of Increases in i Induced by Focal Stimulation in ES Guts
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We found that focal stimulation (5¨C10 Hz, 100 µseconds, 10 pulses) of ES guts differentiated from BDNF  EBs elicited the response of increases in i as reliable evidence for the existence of an enteric neural network in the ES gut (Fig. 5C, 5D), which included a large NF  enteric neural ganglion that was approximately 80 µm in diameter (Fig. 5C) and extensively branching nerve processes (Fig. 5D). In fact, we were able to identify multiple ganglia in ES guts based on NF (Fig. 3A) or PGP 9.5 immunoreactivity (data not shown). Similar observations were made in 17 other ES guts. Consistency of physiological results with immunohistochemical results strongly supported the existence of a physiologically functioning enteric neural network in the ES gut.4 z* H% U) e, }1 b0 a7 E  d' ~1 b
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Figure 4. Increases in i images obtained after focal stimulation. The time courses of the changes in fluo-3 fluorescence at points 1, 2, and 3 are shown in the right traces. iv: Corresponding bright-field image showing the site of the electrode used for focal stimulation of this ES gut. Abbreviations: BDNF, brain-derived neurotrophic factor; EB, embryoid body; ES, embryonic stem; NF, neurofilament., V8 I" l; ~- Z/ Q" `/ o* U: n
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Figure 5. Increases in i elicited by focal stimulation in a dome-like ES gut (A). The response originated in the wall in an ES gut differentiated from the EB (BDNF ) and propagated widely along its edge (from points 1¨C2 and 3 in (A). The time courses of the changes in fluo-3 fluorescence (expressed as Ft/F0) at points 1, 2, and 3 are shown in the lower traces. The response was almost completely abolished by TTX (B). NF immunoreactivity in the same ES gut reveals a large enteric neural ganglion (C) with neural cell processes (D). Abbreviations: BDNF, brain-derived neurotrophic factor; EB, embryoid body; ES, embryonic stem; NF, neurofilament; TTX, tetrodotoxin.4 d. S0 D2 h/ S3 l7 S% h4 \
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Figure 6. Increases in i elicited by focal stimulation (A) were largely attenuated by hexamethonium (B), and the small remaining increases were abolished by TTX (C). Abbreviation: TTX, tetrodotoxin.

We were also able to elicit the response of increases in i response in four ES guts was largely attenuated by hexamethonium (100 µM), which had no effect in one ES gut (Fig. 6B).

Figure 7. Increases in i elicited by focal stimulation at multiple areas (A) were almost completely abolished by atropine (B).

DISCUSSION. z4 Z1 V0 i- s' k
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We previously showed that ES cells can give rise to a functional organ-like unit, the ES gut, which consists of a broad array of enteric derivatives from all three embryonic germ layers, including various kinds of epithelial cells, smooth muscle cells, and ICCs . Thus, we describe here the first successful effort to stimulate differentiation of an ES gut containing enteric neural ganglia; the resultant organ-like cluster exhibited highly coordinated peristalsis-like movements.
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During early hanging-drop culture in the presence of BDNF, trkB, c-ret, sox9, and p75 were expressed. In BDNF  EBs (in late hanging-drop culture), we detected trkB and c-ret, both of which are expressed during in vivo development of enteric neurons during mouse embryogenesis .

Other c-ret ligands, NT-3, and GDNF showed qualitatively similar effects, but those effects on differentiation of enteric neural network structure in the ES gut seemed to be less potent than those of BDNF, although further studies at higher concentrations than the present one are needed. Taken together, these findings suggest a scenario in which BDNF binds to trkB, leading to formation of enteric neural crest cells, which in turn intensely differentiate into enteric neural ganglion cells that form a network able to mediate peristalsis-like motor activity. Notably, this in vitro differentiation process seems reminiscent of ENS development during mouse embryogenesis, when neural crest cells migrate into the GI tract and develop into enteric neurons .

The scenario summarized above is supported by our observation that focal electrical stimulation of ES guts elicited distinct increases in .4 t) W6 r) ^! T% m6 E

Although ETB receptors are briefly required to mediate migration of gut neural crest stem cells .6 Z: T# }  t' J. Q, w( T, ~, j
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CONCLUSION5 V7 g* c1 C; e% O) t# a- W: X

We for the first time have succeeded in combining improved technology (EB culture) with appropriate addition of an exogenous neurotrophic factor to specifically induce differentiation of an ES gut equipped with a functional enteric neuronal network structure ("second brain") mediated via neural-crest formation. This new approach could facilitate significant advances in studies of the integrative physiological functions of the gut and in the development of a complete gut-like organ for regenerative medicine.

DISCLOSURES

The authors indicate no potential conflicts of interest.

ACKNOWLEDGMENTS! w2 P* W2 [: _  x/ J/ [" v$ R# a

This work was supported by grants-in-aid for Scientific Research (14657311, 16650090, and 17300130 for M.T. and 15300134 for S.N.) from the Ministry of Education, Science, Sports and Culture of Japan.: y1 }. O8 _+ o. p
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