Abstract
Stem cell therapy offers the potential of rebuilding the enteric nervous system (ENS) in the aganglionic bowel of patients with Hirschsprung’s disease. P0-Cre/Floxed-EGFP mice in which neural crest-derived cells express EGFP were used to obtain ENS stem/progenitor cells. ENS stem/progenitor cells were transplanted into the bowel of Ret−/− mouse, an animal model of Hirschsprung’s disease. Immunohistochemical analysis was performed to determine whether grafted cells gave rise to neurons in the recipient bowel. EGFP expressing neural crest-derived cells accounted for 7.01 ± 2.52 % of total cells of gastrointestinal tract. ENS stem/progenitor cells were isolated using flow cytometry and expanded as neurosphere-like bodies (NLBs) in a serum-free culture condition. Some cells in NLBs expressed neural crest markers, p75 and Sox10 and neural stem/progenitor cells markers, Nestin and Musashi1. Multipotency of isolated ENS stem/progenitor cells was determined as they differentiated into neurons, glial cells, and myofibloblasts in culture. When co-cultured with explants of hindgut of Ret−/− mice, ENS stem/progenitor cells migrated into the aganglionic bowel and gave rise to neurons. ENS stem/progenitor cells used in this study appear to be clinically relevant donor cells in cell therapy to treat Hirschsprung’s disease capable of colonizing the affected bowel and giving rise to neurons.
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Almond S, Lindley RM, Kenny SE, Connell MG, Edgar DH (2007) Characterisation and transplantation of enteric nervous system progenitor cells. Gut 56:489–496
Azan G, Low WC, Wendelschafer-Crabb G, Ikramuddin S, Kennedy WR (2011) Evidence for neural progenitor cells in the human adult enteric nervous system. Cell Tissue Res 344:217–225
Becker L, Peterson J, Kulkarni S, Pasricha PJ (2013) Ex vivo neurogenesis within enteric ganglia occurs in a PTEN dependent manner. PLoS One 8:e59452
Belkind-Gerson J, Carreon-Rodriguez A, Benedict LA, Steiger C, Pieretti A, Nagy N, Dietrich J, Goldstein AM (2013) Nestin-expressing cells in the gut give rise to enteric neurons and glial cells. Neurogastroenterol Motil 25:61–69
Bondurand N, Natarajan D, Thapar N, Atkins C, Pachnis V (2003) Neuron and glia generating progenitors of the mammalian enteric nervous system isolated from foetal and postnatal gut cultures. Development 130:6387–6400
Bondurand N, Natarajan D, Barlow A, Thapar N, Pachnis V (2006) Maintenance of mammalian enteric nervous system progenitors by SOX10 and endothelin 3 signalling. Development 133:2075–2086
Breau MA, Pietri T, Eder O, Blanche M, Brakebusch C, Fässler R, Thiery JP, Dufour S (2006) Lack of beta1 integrins in enteric neural crest cells leads to a Hirschsprung-like phenotype. Development 133:1725–1734
Catto-Smith AG, Trajanovska M, Taylor RG (2007) Long-term continence after surgery for Hirschsprung’s disease. J Gastroenterol Hepatol 22:2273–2282
Durbec PL, Larsson-Blomberg LB, Schuchardt A, Costantini F, Pachnis V (1996) Common origin and developmental dependence on c-ret of subsets of enteric and sympathetic neuroblasts. Development 122:349–358
Fu M, Vohra BP, Wind D, Heuckeroth RO (2006) BMP signaling regulates murine enteric nervous system precursor migration, neurite fasciculation, and patterning via altered Ncam1 polysialic acid addition. Dev Biol 299:137–150
Furness JB (2012) The enteric nervous system and neurogastroenterology. Nat Rev Gastroenterol Hepatol 9:286–294
Goldstein AM, Brewer KC, Doyle AM, Nagy N, Roberts DJ (2005) BMP signaling is necessary for neural crest cell migration and ganglion formation in the enteric nervous system. Mech Dev 122:821–833
Hearn CJ, Young HM, Ciampoli D, Lomax AE, Newgreen D (1999) Catenary cultures of embryonic gastrointestinal tract support organ morphogenesis, motility, neural crest cell migration, and cell differentiation. Dev Dyn 214:239–247
Hotta R, Stamp L, Foong JP, McConnell SN, Bergner AJ, Anderson RB, Enomoto H, Newgreen DF, Obermayr F, Furness JB, Young HM (2013) Transplanted progenitors generate functional enteric neurons in the postnatal colon. J Clin Invest 123:1182–1191
Ieiri S, Nakatsuji T, Akiyoshi J, Higashi M, Hashizume M, Suita S, Taguchi T (2010) Long-term outcomes and the quality of life of Hirschsprung disease in adolescents who have reached 18 years or older-a 47-year single-institute experience. J Pediatr Surg 45:2398–2402
Joseph NM, He S, Quintana E, Kim YG, Núñez G, Morrison SJ (2011) Enteric glia are multipotent in culture but primarily form glia in the adult rodent gut. J Clin Invest 121:3398–3411
Kaneko Y, Sakakibara S, Imai T, Suzuki A, Nakamura Y, Sawamoto K, Ogawa Y, Toyama Y, Miyata T, Okano H (2000) Musashi1: an evolutionally conserved marker for CNS progenitor cells including neural stem cells. Dev Neurosci 22:139–153
Kawamoto S, Niwa H, Tashiro F, Sano S, Kondoh G, Takeda J, Tabayashi K, Miyazaki J (2000) A novel reporter mouse strain that expresses enhanced green fluorescent protein upon Cre-mediated recombination. FEBS Lett 470:263–268
Kruger G, Mosher J, Bixby S, Joseph N, Iwashita T, Morrison SJ (2002) Neural crest stem cells persist in the adult gut but undergo changes in self-renewal, neuronal subtype potential, and factor responsiveness. Neuron 35:657–669
Laranjeira C, Sandgren K, Kessaris N, Richardson W, Potocnik A, Vanden Berghe P, Pachnis V (2011) Glial cells in the mouse enteric nervous system can undergo neurogenesis in response to injury. J Clin Invest 121:3412–3424
Lee MJ, Calle E, Brennan A, Ahmed S, Sviderskaya E, Jessen KR, Mirsky R (2001) In early development of the rat mRNA for the major myelin protein P(0) is expressed in nonsensory areas of the embryonic inner ear, notochord, enteric nervous system, and olfactory ensheathing cells. Dev Dyn 222:40–51
Lendahl U, Zimmerman LB, McKay RD (1990) CNS stem cells express a new class of intermediate filament protein. Cell 60:585–595
Lindley RM, Hawcutt DB, Connell MG, Almond SL, Vannucchi MG, Faussone-Pellegrini MS, Edgar DH, Kenny SE (2008) Human and mouse enteric nervous system neurosphere transplants regulate the function of aganglionic embryonic distal colon. Gastroenterology 135:205–216
Liu MT, Kuan YH, Wang J et al (2009) 5-HT4 receptor-mediated neuroprotection and neurogenesis in the enteric nervous system of adult mice. J Neurosci 29:9683-9699
Matsuzaki Y, Kinjo K, Mulligan RC, Okano H (2004) Unexpectedly efficient homing capacity of purified murine hematopoietic stem cells. Immunity 20:87–93
Metzger M, Caldwell C, Barlow AJ, Burns AJ, Thapar N (2009) Enteric nervous system stem cells derived from human gut mucosa for the treatment of aganglionic gut disorders. Gastroenterology 136:2214–2225
Mosher JT, Yeager KJ, Kruger GM, Joseph NM, Hutchin ME, Dlugosz AA, Morrison SJ (2007) Intrinsic differences among spatially distinct neural crest stem cells in terms of migratory properties, fate determination, and ability to colonize the enteric nervous system. Dev Biol 303:1–15
Nagoshi N, Shibata S, Kubota Y, Nakamura M, Nagai Y, Satoh E, Morikawa S, Okada Y, Mabuchi Y, Katoh H, Okada S, Fukuda K, Suda T, Matsuzaki Y, Toyama Y, Okano H (2008) Ontogeny and multipotency of neural crest-derived stem cells in mouse bone marrow, dorsal root ganglia, and whisker pad. Cell Stem Cell 2:392–403
Natarajan D, Grigoriou M, Marcos-Gutierrez CV, Atkins C, Pachnis V (1999) Multipotential progenitors of the mammalian enteric nervous system capable of colonising aganglionic bowel in organ culture. Development 126:157–168
Okano H, Imai T, Okabe M (2002) Musashi: a translational regulator of cell fate. J Cell Sci 115:1355–1359
Paratore C, Goerich DE, Suter U, Wegner M, Sommer L (2001) Survival and glial fate acquisition of neural crest cells are regulated by an interplay between the transcription factor Sox10 and extrinsic combinatorial signaling. Development 128:3949–3961
Potten CS, Booth C, Tudor GL, Booth D, Brady G, Hurley P, Ashton G, Clarke R, Sakakibara S, Okano H (2003) Identification of a putative intestinal stem cell and early lineage marker; musashi-1. Differentiation 71:28–41
Reynolds BA, Weiss S (1992) Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255:1707–1710
Sakakibara S, Imai T, Hamaguchi K, Okabe M, Aruga J, Nakajima K, Yasutomi D, Nagata T, Kurihara Y, Uesugi S, Miyata T, Ogawa M, Mikoshiba K, Okano H (1996) Mouse-Musashi-1, a neural RNA-binding protein highly enriched in the mammalian CNS stem cell. Dev Biol 176:230–242
Sawamoto K, Nakao N, Kobayashi K, Matsushita N, Takahashi H, Kakishita K, Yamamoto A, Yoshizaki T, Terashima T, Murakami F, Itakura T, Okano H (2001) Visualization, direct isolation, and transplantation of midbrain dopaminergic neurons. Proc Natl Acad Sci USA 98:6423–6428
Schuchardt A, D’Agati V, Larsson-Blomberg L, Costantini F, Pachnis V (1994) Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret. Nature 367:380–383
Stemple DL, Anderson DJ (1992) Isolation of a stem cell for neurons and glia from the mammalian neural crest. Cell 71:973–985
Suarez-Rodriguez R, Belkind-Gerson J (2004) Cultured nestin-positive cells from postnatal mouse small bowel differentiate ex vivo into neurons, glia, and smooth muscle. Stem Cells 22:1373–1385
Sun NF, Zhong WY, Lu SA, Tian YL, Chen JB, Hu SY, Tian AL (2013) Coexpression of recombinant adenovirus carrying GDNF and EDNRB genes in neural stem cells in vitro. Cell Biol Int 37:458–463
Tomita Y, Matsumura K, Wakamatsu Y, Matsuzaki Y, Shibuya I, Kawaguchi H, Ieda M, Kanakubo S, Shimazaki T, Ogawa S, Osumi N, Okano H, Fukuda K (2005) Cardiac neural crest cells contribute to the dormant multipotent stem cell in the mammalian heart. J Cell Biol 170:1135–1146
Yamauchi Y, Abe K, Mantani A, Hitoshi Y, Suzuki M, Osuzu F, Kuratani S, Yamamura K (1999) A novel transgenic technique that allows specific marking of the neural crest cell lineage in mice. Dev Biol 212:191–203
Yoshida S, Shimmura S, Nagoshi N, Fukuda K, Matsuzaki Y, Okano H, Tsubota K (2006) Isolation of multipotent neural crest-derived stem cells from the adult mouse cornea. Stem Cells 24:2714–2722
Young HM, Bergner AJ, Anderson RB, Enomoto H, Milbrandt J, Newgreen DF, Whitington PM (2004) Dynamics of neural crest-derived cell migration in the embryonic mouse gut. Dev Biol 270:455–473
Acknowledgments
We thank M Mori, T Harada, S Suzuki (FACS technician) for technical supports and animal care, M Jijiwa for information about Ret−/− mice, HM Young for helpful discussion to prepare manuscript, M Takahashi for providing Ret−/− mice, F Costantini for giving a permission to have a Ret−/− mice. This work was supported by the Ministry of Education, Science, and Culture of Japan (MEXT), KAKENHI, Grant-in-Aid for Scientific Research (C) 20592091 and Grant-in-Aid for Young Scientists (B) 21791736, by a Grant-in-aid from the Global COE program of MEXT to Keio University.
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Ryuhei Nishikawa and Ryo Hotta equally contributed to this work.
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Nishikawa, R., Hotta, R., Shimojima, N. et al. Migration and differentiation of transplanted enteric neural crest-derived cells in murine model of Hirschsprung’s disease. Cytotechnology 67, 661–670 (2015). https://doi.org/10.1007/s10616-014-9754-8
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DOI: https://doi.org/10.1007/s10616-014-9754-8