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Future Aspect

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Abstract

Overall outcome of Hirschsprung disease (HSCR) remain inadequate, and the treatment options for severe forms are limited to palliative interventions. Advances in molecular biology and genetics have led to development of cellular therapy for a potential curative treatment for HSCR. The field of cell therapies for HSCR has seen tremendous progress in recent years. A variety of sources for neural progenitor cells have been identified ranging from adult tissue-derived cells (endogenous enteric nervous system (ENS)-derived enteric neural progenitors (ENPCs)) to pluripotent stem cells (embryonic stem (ES) cells and induced pluripotent stem (iPS) cells). Key recent advances include reports of the efficient derivation and cultivation of ENPCs from either endogenous ENS or pluripotent stem cells and their further differentiation into functional enteric neurons in vitro. Following successful transplantation of these ENPCs into colon of mice with enteric neuropathies, including HSCR in vivo, functional integration into host enteric circuitry and rescued mortality of recipient mice have been achieved. A number of key challenges remain, however, before effective clinical application. These include the need to better understand the condition of aganglionic colon to accept transplanted cells, to optimize/manipulate cellular potential to maximize the success of cellular treatment, and to determine the long-term safety and efficacy of cell therapy. Nonetheless, the impressive recent progress raises further hope for the imminent use of cell transplantation as an effective therapy for HSCR.

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References

  1. Furness JB. The enteric nervous system. Hoboken, NJ: Blackwell Publishing; 2006.

    Google Scholar 

  2. Heanue TA, Pachnis V. Enteric nervous system development and Hirschsprung’s disease: advances in genetic and stem cell studies. Nat Rev Neurosci. 2007;8(6):466–79.

    Article  CAS  PubMed  Google Scholar 

  3. Obermayr F, Hotta R, Enomoto H, Young HM. Development and developmental disorders of the enteric nervous system. Nat Rev Gastroenterol Hepatol. 2013;10(1):43–57.

    Article  CAS  PubMed  Google Scholar 

  4. Goldstein AM, Hofstra RM, Burns AJ. Building a brain in the gut: development of the enteric nervous system. Clin Genet. 2013;83(4):307–16.

    Article  CAS  PubMed  Google Scholar 

  5. Hotta R, Natarajan D, Thapar N. Potential of cell therapy to treat pediatric motility disorders. Semin Pediatr Surg. 2009;18(4):263–73.

    Article  PubMed  Google Scholar 

  6. Stamp LA. Cell therapy for Gi motility disorders: comparison of cell sources and proposed steps for treating hirschsprung disease. Am J Physiol Gastrointest Liver Physiol. 2017;312(4):G348–54.

    Article  PubMed  Google Scholar 

  7. Burns AJ, Thapar N. Neural stem cell therapies for enteric nervous system disorders. Nat Rev Gastroenterol Hepatol. 2014;11(5):317–28.

    Article  PubMed  Google Scholar 

  8. Burns AJ, Goldstein AM, Newgreen DF, Stamp L, Schafer KH, Metzger M, et al. White paper on guidelines concerning enteric nervous system stem cell therapy for enteric neuropathies. Dev Biol. 2016;417(2):229–51.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Duncan T, Valenzuela M. Alzheimer’s disease, dementia, and stem cell therapy. Stem Cell Res Ther. 2017;8(1):111.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Goldman SA. Stem and progenitor cell-based therapy of the central nervous system: hopes, hype, and wishful thinking. Cell Stem Cell. 2016;18(2):174–88.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Kulkarni S, Becker L, Pasricha PJ. Stem cell transplantation in neurodegenerative disorders of the gastrointestinal tract: future or fiction? Gut. 2012;61(4):613–21.

    Article  CAS  PubMed  Google Scholar 

  12. Farlie PG, McKeown SJ, Newgreen DF. The neural crest: basic biology and clinical relationships in the craniofacial and enteric nervous systems. Birth Defects Res C Embryo Today. 2004;72(2):173–89.

    Article  CAS  PubMed  Google Scholar 

  13. Le Douarin NM, Kalcheim C. The neural crest. Cambridge: Cambridge University Press; 1999.

    Book  Google Scholar 

  14. Young HM, Newgreen DF, Burns AJ. Development of the enteric nervous system in relation to Hirschsprung’s disease. In: Ferretti P, Copp A, Tickle C, Moore G, editors. Embryos, genes and birth defects. Hoboken, NJ: Wiley; 2004. p. 263–300.

    Google Scholar 

  15. Kruger G, Mosher J, Bixby S, Joseph N, Iwashita T, Morrison S. Neural crest stem cells persist in the adult gut but undergo changes in self-renewal, neuronal subtype potential, and factor responsiveness. Neuron. 2002;35(4):657–69.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Lo L, Anderson DJ. Postmigratory neural crest cells expressing c-RET display restricted developmental and proliferative capacities. Neuron. 1995;15(3):527–39.

    Article  CAS  PubMed  Google Scholar 

  17. Morrison SJ, White PM, Zock C, Anderson DJ. Prospective identification, isolation by flow cytometry, and in vivo self-renewal of multipotent mammalian neural crest stem cells. Cell. 1999 Mar 5;96(5):737–49.

    Article  CAS  PubMed  Google Scholar 

  18. Natarajan D, Grigoriou M, Marcos-Gutierrez CV, Atkins C, Pachnis V. Multipotential progenitors of the mammalian enteric nervous system capable of colonising aganglionic bowel in organ culture. Development. 1999;126(1):157–68.

    CAS  PubMed  Google Scholar 

  19. Sidebotham EL, Kenny SE, Lloyd DA, Vaillant CR, Edgar DH. Location of stem cells for the enteric nervous system. Pediatr Surg Int. 2002;18(7):581–5.

    Article  CAS  PubMed  Google Scholar 

  20. Almond S, Lindley RM, Kenny SE, Connell MG, Edgar DH. Characterisation and transplantation of enteric nervous system progenitor cells. Gut. 2007;56(4):489–96.

    Article  PubMed  Google Scholar 

  21. Bondurand N, Natarajan D, Thapar N, Atkins C, Pachnis V. Neuron and glia generating progenitors of the mammalian enteric nervous system isolated from foetal and postnatal gut cultures. Development. 2003;130(25):6387–400.

    Article  CAS  PubMed  Google Scholar 

  22. Metzger M, Bareiss PM, Danker T, Wagner S, Hennenlotter J, Guenther E, et al. Expansion and differentiation of neural progenitors derived from the human adult enteric nervous system. Gastroenterology. 2009;137(6):2063–73.. e4

    Article  CAS  PubMed  Google Scholar 

  23. Hotta R, Stamp LA, Foong JP, McConnell SN, Bergner AJ, Anderson RB, et al. Transplanted progenitors generate functional enteric neurons in the postnatal colon. J Clin Invest. 2013;123(3):1182–91.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Tsai YH, Murakami N, Gariepy CE. Postnatal intestinal engraftment of prospectively selected enteric neural crest stem cells in a rat model of Hirschsprung disease. Neurogastroenterol Motil. 2011;23(4):362–9.

    Article  PubMed  Google Scholar 

  25. Dettmann HM, Zhang Y, Wronna N, Kraushaar U, Guenther E, Mohr R, et al. Isolation, expansion and transplantation of postnatal murine progenitor cells of the enteric nervous system. PLoS One. 2014;9(5):e97792.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. Natarajan D, Cooper J, Choudhury S, Delalande JM, McCann C, Howe SJ, et al. Lentiviral labeling of mouse and human enteric nervous system stem cells for regenerative medicine studies. Neurogastroenterol Motil. 2014;26(10):1513–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Binder E, Natarajan D, Cooper J, Kronfli R, Cananzi M, Delalande JM, et al. Enteric neurospheres are not specific to neural crest cultures: implications for neural stem cell therapies. PLoS One. 2015;10(3):e0119467.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Cheng LS, Hotta R, Graham HK, Nagy N, Goldstein AM, Belkind-Gerson J. Endoscopic delivery of enteric neural stem cells to treat Hirschsprung disease. Neurogastroenterol Motil. 2015;27(10):1509–14.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Hotta R, Cheng LS, Graham HK, Pan W, Nagy N, Belkind-Gerson J, et al. Isogenic enteric neural progenitor cells can replace missing neurons and glia in mice with Hirschsprung disease. Neurogastroenterol Motil. 2016;28(4):498–512.

    Article  CAS  PubMed  Google Scholar 

  30. McCann CJ, Cooper JE, Natarajan D, Jevans B, Burnett LE, Burns AJ, et al. Transplantation of enteric nervous system stem cells rescues nitric oxide synthase deficient mouse colon. Nat Commun. 2017;8:15937.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Stamp LA, Gwynne RM, Foong JP, Lomax AE, Hao MM, Kaplan DI, et al. Optogenetic demonstration of functional innervation of mouse colon by neurons derived from transplanted neural cells. Gastroenterology. 2017;152(6):1407–18.

    Article  PubMed  Google Scholar 

  32. Cooper JE, McCann CJ, Natarajan D, Choudhury S, Boesmans W, Delalande JM, et al. In vivo transplantation of enteric neural crest cells into mouse gut; engraftment, functional integration and long-term safety. PLoS One. 2016;11(1):e0147989.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Rauch U, Hansgen A, Hagl C, Holland-Cunz S, Schafer KH. Isolation and cultivation of neuronal precursor cells from the developing human enteric nervous system as a tool for cell therapy in dysganglionosis. Int J Color Dis. 2006;21(6):554–9.

    Article  Google Scholar 

  34. Lindley RM, Hawcutt DB, Connell MG, Almond SN, Vannucchi MG, Faussone-Pellegrini MS, et al. Human and mouse enteric nervous system neurosphere transplants regulate the function of aganglionic embryonic distal colon. Gastroenterology. 2008;135(1):205–16.

    Article  PubMed  Google Scholar 

  35. Metzger M, Caldwell C, Barlow AJ, Burns AJ, Thapar N. Enteric nervous system stem cells derived from human gut mucosa for the treatment of aganglionic gut disorders. Gastroenterology. 2009;136(7):2214–25.

    Article  CAS  PubMed  Google Scholar 

  36. Hetz S, Acikgoez A, Voss U, Nieber K, Holland H, Hegewald C, et al. In vivo transplantation of neurosphere-like bodies derived from the human postnatal and adult enteric nervous system: a pilot study. PLoS One. 2014;9(4):e93605.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. Rollo BN, Zhang D, Stamp LA, Menheniott TR, Stathopoulos L, Denham M, et al. Enteric neural cells from hirschsprung disease patients form ganglia in autologous aneuronal colon. Cell Mol Gastroenterol Hepatol. 2016;2(1):92–109.

    Article  PubMed  Google Scholar 

  38. Cooper JE, Natarajan D, McCann CJ, Choudhury S, Godwin H, Burns AJ, et al. In vivo transplantation of fetal human gut-derived enteric neural crest cells. Neurogastroenterol Motil. 2017;29(1).

    Google Scholar 

  39. Cheng LS, Hotta R, Graham HK, Belkind-Gerson J, Nagy N, Goldstein AM. Postnatal human enteric neuronal progenitors can migrate, differentiate, and proliferate in embryonic and postnatal aganglionic gut environments. Pediatr Res. 2017;81(5):838–46.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, et al. Embryonic stem cell lines derived from human blastocysts. Science. 1998;282(5391):1145–7.

    Article  CAS  PubMed  Google Scholar 

  41. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126(4):663–76.

    Article  CAS  PubMed  Google Scholar 

  42. Hotta R, Pepdjonovic L, Anderson RB, Zhang D, Bergner AJ, Leung J, et al. Small-molecule induction of neural crest-like cells derived from human neural progenitors. Stem Cells. 2009;27(12):2896–905.

    CAS  PubMed  Google Scholar 

  43. Kawaguchi J, Nichols J, Gierl MS, Faial T, Smith A. Isolation and propagation of enteric neural crest progenitor cells from mouse embryonic stem cells and embryos. Development. 2010;137(5):693–704.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Sasselli V, Micci MA, Kahrig KM, Pasricha PJ. Evaluation of ES-derived neural progenitors as a potential source for cell replacement therapy in the gut. BMC Gastroenterol. 2012;12:81.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Fattahi F, Steinbeck JA, Kriks S, Tchieu J, Zimmer B, Kishinevsky S, et al. Deriving human ENS lineages for cell therapy and drug discovery in Hirschsprung disease. Nature. 2016;531(7592):105–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Li W, Huang L, Zeng J, Lin W, Li K, Sun J, et al. Characterization and transplantation of enteric neural crest cells from human induced pluripotent stem cells. Mol Psychiatry. 2016;23(3):499–508.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. Wobus AM, Boheler KR. Embryonic stem cells: prospects for developmental biology and cell therapy. Physiol Rev. 2005;85(2):635–78.

    Article  CAS  PubMed  Google Scholar 

  48. Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature. 1981;292(5819):154–6.

    Article  CAS  PubMed  Google Scholar 

  49. Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci U S A. 1981;78(12):7634–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131(5):861–72.

    Article  CAS  PubMed  Google Scholar 

  51. Lee G, Chambers SM, Tomishima MJ, Studer L. Derivation of neural crest cells from human pluripotent stem cells. Nat Protoc. 2010;5(4):688–701.

    Article  CAS  PubMed  Google Scholar 

  52. Jiang X, Gwye Y, McKeown SJ, Bronner-Fraser M, Lutzko C, Lawlor ER. Isolation and characterization of neural crest stem cells derived from in vitro-differentiated human embryonic stem cells. Stem Cells Dev. 2009;18(7):1059–70.

    Article  CAS  PubMed  Google Scholar 

  53. Mica Y, Lee G, Chambers SM, Tomishima MJ, Studer L. Modeling neural crest induction, melanocyte specification, and disease-related pigmentation defects in hESCs and patient-specific iPSCs. Cell Rep. 2013;3(4):1140–52.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Young HM, Ciampoli D, Southwell BR, Newgreen DF. Origin of interstitial cells of Cajal in the mouse intestine. Dev Biol. 1996;180(1):97–107.

    Article  CAS  PubMed  Google Scholar 

  55. Saffrey MJ. Cellular changes in the enteric nervous system during ageing. Dev Biol. 2013;382(1):344–55.

    Article  CAS  PubMed  Google Scholar 

  56. Barlow AJ, Wallace AS, Thapar N, Burns AJ. Critical numbers of neural crest cells are required in the pathways from the neural tube to the foregut to ensure complete enteric nervous system formation. Development. 2008;135(9):1681–91.

    Article  CAS  PubMed  Google Scholar 

  57. Young HM, Bergner AJ, Anderson RB, Enomoto H, Milbrandt J, Newgreen DF, et al. Dynamics of neural crest-derived cell migration in the embryonic mouse gut. Dev Biol. 2004;270(2):455–73.

    Article  CAS  PubMed  Google Scholar 

  58. Hotta R, Cheng LS, Graham HK, Nagy N, Belkind-Gerson J, Mattheolabakis G, et al. Delivery of enteric neural progenitors with 5-HT4 agonist-loaded nanoparticles and thermosensitive hydrogel enhances cell proliferation and differentiation following transplantation in vivo. Biomaterials. 2016;88:1–11.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. McKeown SJ, Mohsenipour M, Bergner AJ, Young HM, Stamp LA. Exposure to GDNF enhances the ability of enteric neural progenitors to generate an enteric nervous system. Stem Cell Reports. 2017;8(2):476–88.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Martucciello G, Thompson H, Mazzola C, Morando A, Bertagnon M, Negri F, et al. GDNF deficit in Hirschsprung’s disease. J Pediatr Surg. 1998;33(1):99–102.

    Article  CAS  PubMed  Google Scholar 

  61. Lui VC, Samy ET, Sham MH, Mulligan LM, Tam PK. Glial cell line-derived neurotrophic factor family receptors are abnormally expressed in aganglionic bowel of a subpopulation of patients with Hirschsprung’s disease. Lab Investig. 2002;82(6):703–12.

    Article  CAS  PubMed  Google Scholar 

  62. Pierre JF, Barlow-Anacker AJ, Erickson CS, Heneghan AF, Leverson GE, Dowd SE, et al. Intestinal dysbiosis and bacterial enteroinvasion in a murine model of Hirschsprung’s disease. J Pediatr Surg. 2014;49(8):1242–51.

    Article  PubMed  PubMed Central  Google Scholar 

  63. Frykman PK, Nordenskjold A, Kawaguchi A, Hui TT, Granstrom AL, Cheng Z, et al. Characterization of bacterial and fungal microbiome in children with Hirschsprung disease with and without a history of enterocolitis: a multicenter study. PLoS One. 2015;10(4):e0124172.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  64. Micci MA, Kahrig KM, Simmons RS, Sarna SK, Espejo-Navarro MR, Pasricha PJ. Neural stem cell transplantation in the stomach rescues gastric function in neuronal nitric oxide synthase-deficient mice. Gastroenterology. 2005;129(6):1817–24.

    Article  CAS  PubMed  Google Scholar 

  65. Zhou YY, Zeng F. Integration-free methods for generating induced pluripotent stem cells. Genomics Proteomics Bioinformatics. 2013;11(5):284–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Iwashita T, Kruger GM, Pardal R, Kiel MJ, Morrison SJ. Hirschsprung disease is linked to defects in neural crest stem cell function. Science. 2003;301(5635):972–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Lai FP, Lau ST, Wong JK, Gui H, Wang RX, Zhou T, et al. Correction of Hirschsprung-associated mutations in human induced pluripotent stem cells, via CRISPR/Cas9, restores neural crest cell function. Gastroenterology. 2017;153(1):139–153.e8.

    Article  CAS  PubMed  Google Scholar 

  68. Seki T, Yuasa S, Oda M, Egashira T, Yae K, Kusumoto D, et al. Generation of induced pluripotent stem cells from human terminally differentiated circulating T cells. Cell Stem Cell. 2010;7(1):11–4.

    Article  CAS  PubMed  Google Scholar 

  69. Park HY, Noh EH, Chung HM, Kang MJ, Kim EY, Park SP. Efficient generation of virus-free iPS cells using liposomal magnetofection. PLoS One. 2012;7(9):e45812.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Takaki M, Nakayama S, Misawa H, Nakagawa T, Kuniyasu H. In vitro formation of enteric neural network structure in a gut-like organ differentiated from mouse embryonic stem cells. Stem Cells. 2006;24(6):1414–22.

    Article  CAS  PubMed  Google Scholar 

  71. Ueda T, Yamada T, Hokuto D, Koyama F, Kasuda S, Kanehiro H, et al. Generation of functional gut-like organ from mouse induced pluripotent stem cells. Biochem Biophys Res Commun. 2010;391(1):38–42.

    Article  CAS  PubMed  Google Scholar 

  72. Kitano K, Schwartz DM, Zhou H, Gilpin SE, Wojtkiewicz GR, Ren X, et al. Bioengineering of functional human induced pluripotent stem cell-derived intestinal grafts. Nat Commun. 2017;8(1):765.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  73. Workman MJ, Mahe MM, Trisno S, Poling HM, Watson CL, Sundaram N, et al. Engineered human pluripotent-stem-cell-derived intestinal tissues with a functional enteric nervous system. Nat Med. 2017;23(1):49–59.

    Article  CAS  PubMed  Google Scholar 

  74. Schlieve CR, Fowler KL, Thornton M, Huang S, Hajjali I, Hou X, et al. Neural crest cell implantation restores enteric nervous system function and alters the gastrointestinal transcriptome in human tissue-engineered small intestine. Stem Cell Reports. 2017;9(3):883–96.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Hanna J, Wernig M, Markoulaki S, Sun CW, Meissner A, Cassady JP, et al. Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science. 2007;318(5858):1920–3.

    Article  CAS  PubMed  Google Scholar 

  76. Hallett PJ, Deleidi M, Astradsson A, Smith GA, Cooper O, Osborn TM, et al. Successful function of autologous iPSC-derived dopamine neurons following transplantation in a non-human primate model of Parkinson’s disease. Cell Stem Cell. 2015;16(3):269–74.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Lindley RM, Hawcutt DB, Connell MG, Edgar DH, Kenny SE. Properties of secondary and tertiary human enteric nervous system neurospheres. J Pediatr Surg. 2009;44(6):1249–55. discussion 55-6.

    Article  PubMed  Google Scholar 

  78. Reubinoff BE, Itsykson P, Turetsky T, Pera MF, Reinhartz E, Itzik A, et al. Neural progenitors from human embryonic stem cells. Nat Biotechnol. 2001;19(12):1134–40.

    Article  CAS  PubMed  Google Scholar 

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Bhave, S., Hotta, R. (2019). Future Aspect. In: Taguchi, T., Matsufuji, H., Ieiri, S. (eds) Hirschsprung’s Disease and the Allied Disorders. Springer, Singapore. https://doi.org/10.1007/978-981-13-3606-5_31

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