Pediatric Surgery International

, Volume 28, Issue 2, pp 135–142 | Cite as

Smooth muscle proteins from Hirschsprung’s disease facilitates stem cell differentiation

  • Cornelia Irene Hagl
  • Sabine Heumüller
  • Markus Klotz
  • Ulrike Subotic
  • Lucas Wessel
  • Karl-Herbert SchäferEmail author
Original Article


Background and aims

The transplantation of neural crest derived stem cells (NCSC’s) is a potent alternative for the treatment of Hirschsprung’s disease (HSCR). Cells to be transplanted should find an appropriate microenvironment to survive and differentiate. To investigate the quality of this microenvironment, effects of HSCR-smooth-muscle-protein extracts upon NCSC’s were studied in vitro.


Postnatal human gut from children undergoing colonic resection due to HSCR was divided in segments. Smooth muscle was dissected and homogenized. Glial-cell-line-derived-neurotrophic-factor (GDNF) concentration was measured in the homogenates from the individual segment using ELISA. NCSC’s were exposed to protein extracts derived from ganglionic and aganglionic HSCR segments, and their effect upon neurite outgrowth, survival and branching was evaluated.


The amount of the factors varied considerably between the proximal and distal segments, and also from patient to patient. While extracts from proximal segments tended to have more prominent effects, all HSCR-muscle-protein extracts increased neuronal survival and network formation.


Muscle protein from aganglionic bowel supports the survival and outgrowth of NCSC’s and is so an appropriate target for neural stem cell treatment.


Neural crest stem cells (NCSC’s) Enteric nervous system (ENS) Hirschsprung’s disease (HSCR) GDNF 



We are grateful to Elvira Wink and Karin Kaiser for excellent technical support. The work was supported by the German Research Foundation (DFG SCHA 878-1 and 2).


  1. 1.
    Tsuji H, Spitz L, Kiely EM, Drake DP, Pierro A (1999) Management and long-term follow-up of infants with total colonic aganglionosis. J Pediatr Surg 34(1):158–161. pii:S0022-3468(99)90248-8 (discussion 162)Google Scholar
  2. 2.
    Amiel J, Lyonnet S (2001) Hirschsprung disease, associated syndromes, and genetics: a review. J Med Genet 38(11):729–739PubMedCrossRefGoogle Scholar
  3. 3.
    Catto-Smith AG, Trajanovska M, Taylor RG (2007) Long-term continence after surgery for Hirschsprung’s disease. J Gastroenterol Hepatol 22(12):2273–2282. doi: 10.1111/j.1440-1746.2006.04750.x PubMedCrossRefGoogle Scholar
  4. 4.
    Sandgren K, Ekblad E, Larsson LT (2000) Survival of neurons and interstitial cells of Cajal after autotransplantation of myenteric ganglia from small intestine in the lethal spotted mouse. Pediatr Surg Int 16(4):272–276PubMedCrossRefGoogle Scholar
  5. 5.
    Schafer KH, Mestres P (2000) Reaggregation of rat dissociated myenteric plexus in extracellular matrix gels. Dig Dis Sci 45(8):1631–1638PubMedCrossRefGoogle Scholar
  6. 6.
    Schafer KH, Van Ginneken C, Copray S (2009) Plasticity and neural stem cells in the enteric nervous system. Anat Rec (Hoboken) 292(12):1940–1952. doi: 10.1002/ar.21033 CrossRefGoogle Scholar
  7. 7.
    Sidebotham EL, Woodward MN, Kenny SE, Lloyd DA, Vaillant CR, Edgar DH (2002) Localization and endothelin-3 dependence of stem cells of the enteric nervous system in the embryonic colon. J Pediatr Surg 37(2):145–150. pii:S0022346802194188Google Scholar
  8. 8.
    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(25):6387–6400. doi: 10.1242/dev.00857 PubMedCrossRefGoogle Scholar
  9. 9.
    Schafer KH, Hagl CI, Rauch U (2003) Differentiation of neurospheres from the enteric nervous system. Pediatr Surg Int 19(5):340–344. doi: 10.1007/s00383-003-1007-4 PubMedCrossRefGoogle Scholar
  10. 10.
    Schafer KH, Saffrey MJ, Burnstock G, Mestres-Ventura P (1997) A new method for the isolation of myenteric plexus from the newborn rat gastrointestinal tract. Brain Res Brain Res Protoc 1(2):109–113. pii:S1385-299X(96)00017-7Google Scholar
  11. 11.
    Schafer KH, Mestres P (1997) Human newborn and adult myenteric plexus grows in different patterns. Cell Mol Biol (Noisy-le-grand) 43(8):1171–1180Google Scholar
  12. 12.
    Rauch U, Hansgen A, Hagl C, Holland-Cunz S, Schafer KH (2006) Isolation and cultivation of neuronal precursor cells from the developing human enteric nervous system as a tool for cell therapy in dysganglionosis. Int J Colorectal Dis 21(6):554–559. doi: 10.1007/s00384-005-0051-z PubMedCrossRefGoogle Scholar
  13. 13.
    Berrebi D, Fouquet V, de Lagausie P, Carricaburu E, Ferkdadji L, Chomette P, Enezian G, Ezzahir N, Peuchmaur M, Aigrain Y (2007) Duhamel operation vs neonatal transanal endorectal pull-through procedure for Hirschsprung disease: which are the changes for pathologists? J Pediatr Surg 42(4):688–691. doi: 10.1016/J.Jpedsurg.2006.12.015 PubMedCrossRefGoogle Scholar
  14. 14.
    Wang L, Wang ZH, Shen CY, et al (2010) Differentiation of human bone marrow mesenchymal stem cells grown in terpolyesters of 3-hydroxyalkanoates scaffolds into nerve cells. Biomaterials 31:1691–1698Google Scholar
  15. 15.
    Kramer F, Stover T, Warnecke A, et al (2010) BDNF mRNA expression is significantly upregulated in vestibular schwannomas and correlates with proliferative activity. J Neurooncol 98:31–39Google Scholar
  16. 16.
    Bottner M, Bar F, Von Koschitzky H, et al (2010) Laser microdissection as a new tool to investigate site-specific gene expression in enteric ganglia of the human intestine. Neurogastroenterol Motil 22:168–172, e52Google Scholar
  17. 17.
    Barlow AJ, Wallace AS, Thapar N, Burns AJ (2008) 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 135(9):1681–1691. doi: 10.1242/dev.017418 PubMedCrossRefGoogle Scholar
  18. 18.
    Barlow A, de Graaff E, Pachnis V (2003) Enteric nervous system progenitors are coordinately controlled by the G protein-coupled receptor EDNRB and the receptor tyrosine kinase RET. Neuron 40(5):905–916. pii:S089662730300730XGoogle Scholar
  19. 19.
    Furness J (2006) The enteric nervous system. Blackwell, OxfordGoogle Scholar
  20. 20.
    Burzynski G, Shepherd IT, Enomoto H (2009) Genetic model system studies of the development of the enteric nervous system, gut motility and Hirschsprung’s disease. Neurogastroent Motil 21(2):113–127. doi: 10.1111/J.1365-2982.2008.01256.X CrossRefGoogle Scholar
  21. 21.
    Emison ES, Garcia-Barcelo M, Grice EA, Lantieri F, Amiel J, Burzynski G, Fernandez RM, Hao L, Kashuk C, West K, Miao X, Tam PK, Griseri P, Ceccherini I, Pelet A, Jannot AS, de Pontual L, Henrion-Caude A, Lyonnet S, Verheij JB, Hofstra RM, Antinolo G, Borrego S, McCallion AS, Chakravarti A (2010) Differential contributions of rare and common, coding and noncoding Ret mutations to multifactorial Hirschsprung disease liability. Am J Hum Genet 87(1):60–74. doi: 10.1016/j.ajhg.2010.06.007 PubMedCrossRefGoogle Scholar
  22. 22.
    Menezes M, Puri P (2006) Long-term outcome of patients with enterocolitis complicating Hirschsprung’s disease. Pediatr Surg Int 22(4):316–318. doi: 10.1007/s00383-006-1639-2 PubMedCrossRefGoogle Scholar
  23. 23.
    Schafer KH, Micci MA, Pasricha PJ (2009) Neural stem cell transplantation in the enteric nervous system: roadmaps and roadblocks. Neurogastroenterol Motil 21(2):103–112. doi: 10.1111/j.1365-2982.2008.01257.x PubMedCrossRefGoogle Scholar
  24. 24.
    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(7):2214–2225, e2211–e2213. doi: 10.1053/j.gastro.2009.02.048 Google Scholar
  25. 25.
    Hotta R, Natarajan D, Thapar N (2009) Potential of cell therapy to treat pediatric motility disorders. SEmin Pediatr Surg 18(4):263–273. doi: 10.1053/j.sempedsurg.2009.07.008 PubMedCrossRefGoogle Scholar
  26. 26.
    Kruger GM, Mosher JT, 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(4):657–669. pii:S0896627302008279Google Scholar
  27. 27.
    Micci MA, Pasricha PJ (2007) Neural stem cells for the treatment of disorders of the enteric nervous system: strategies and challenges. Dev Dyn 236(1):33–43. doi: 10.1002/dvdy.20975 PubMedCrossRefGoogle Scholar
  28. 28.
    Thapar N (2007) Future horizons in the treatment of enteric neuropathies. J Pediatr Gastroenterol Nutr45 (Suppl 2):S110–S114. doi: 10.1097/MPG.0b013e31812e667c
  29. 29.
    Iwashita T, Kruger GM, Pardal R, Kiel MJ, Morrison SJ (2003) Hirschsprung disease is linked to defects in neural crest stem cell function. Science 301(5635):972–976. doi: 10.1126/science.1085649 PubMedCrossRefGoogle Scholar
  30. 30.
    Paran TS, Rolle U, Puri P (2006) Enteric nervous system and developmental abnormalities in childhood. Pediatr Surg Int 22(12):945–959. doi: 10.1007/s00383-006-1782-9 PubMedCrossRefGoogle Scholar
  31. 31.
    Heanue TA, Pachnis V (2007) Enteric nervous system development and Hirschsprung’s disease: advances in genetic and stem cell studies. Nat Rev Neurosci 8(6):466–479. doi: 10.1038/nrn2137 PubMedCrossRefGoogle Scholar
  32. 32.
    Parikh DH, Tam PK, Van Velzen D, Edgar D (1992) Abnormalities in the distribution of laminin and collagen type IV in Hirschsprung’s disease. Gastroenterology 102(4 Pt 1):1236–1241. pii:S0016508592001550Google Scholar
  33. 33.
    Parikh DH, Tam PK, Van Velzen D, Edgar D (1994) The extracellular matrix components, tenascin and fibronectin, in Hirschsprung’s disease: an immunohistochemical study. J Pediatr Surg 29(10):1302–1306. pii:0022-3468(94)90101-5Google Scholar
  34. 34.
    Rauch U, Schafer KH (2003) The extracellular matrix and its role in cell migration and development of the enteric nervous system. Eur J Pediatr Surg 13(3):158–162. doi: 10.1055/s-2003-41265 PubMedCrossRefGoogle Scholar
  35. 35.
    Rothman TP, Goldowitz D, Gershon MD (1993) Inhibition of migration of neural crest-derived cells by the abnormal mesenchyme of the presumptive aganglionic bowel of ls/ls mice: analysis with aggregation and interspecies chimeras. Dev Biol 159(2):559–573. doi: 10.1006/dbio.1993.1264 PubMedCrossRefGoogle Scholar
  36. 36.
    Jacobs-Cohen RJ, Payette RF, Gershon MD, Rothman TP (1987) Inability of neural crest cells to colonize the presumptive aganglionic bowel of ls/ls mutant mice: requirement for a permissive microenvironment. J Comp Neurol 255(3):425–438. doi: 10.1002/cne.902550309 PubMedCrossRefGoogle Scholar
  37. 37.
    Martucciello G, Brizzolara A, Favre A, Lombardi L, Bocciardi R, Sanguineti M, Pini Prato A, Jasonni V (2007) Neural crest neuroblasts can colonise aganglionic and ganglionic gut in vivo. Eur J Pediatr Surg 17(1):34–40. doi: 10.1055/s-2007-964952 PubMedCrossRefGoogle Scholar
  38. 38.
    Ohshiro K, Puri P (1998) Reduced glial cell line-derived neurotrophic factor level in aganglionic bowel in Hirschsprung’s disease. J Pediatr Surg 33(6):904–908. pii:S0022-3468(98)90671-6Google Scholar
  39. 39.
    Lui VC, Samy ET, Sham MH, Mulligan LM, Tam PK (2002) Glial cell line-derived neurotrophic factor family receptors are abnormally expressed in aganglionic bowel of a subpopulation of patients with Hirschsprung’s disease. Lab Invest 82(6):703–712PubMedGoogle Scholar
  40. 40.
    Rodrigues DM, Li AY, Nair DG, Blennerhassett MG (2010) Glial cell line-derived neurotrophic factor is a key neurotrophin in the postnatal enteric nervous system. Neurogastroenterol Motil. doi: 10.1111/j.1365-2982.2010.01626.x
  41. 41.
    Young HM, Hearn CJ, Farlie PG, Canty AJ, Thomas PQ, Newgreen DF (2001) GDNF is a chemoattractant for enteric neural cells. Dev Biol 229(2):503–516. 10.1006/dbio.2000.0100S0012-1606(00)90100-3[pii]PubMedCrossRefGoogle Scholar
  42. 42.
    Krieglstein K, Henheik P, Farkas L, Jaszai J, Galter D, Krohn K, Unsicker K (1998) Glial cell line-derived neurotrophic factor requires transforming growth factor-beta for exerting its full neurotrophic potential on peripheral and CNS neurons. J Neurosci 18(23):9822–9834PubMedGoogle Scholar
  43. 43.
    Peterziel H, Paech T, Strelau J, Unsicker K, Krieglstein K (2007) Specificity in the crosstalk of TGF beta/GDNF family members is determined by distinct GFR alpha receptors. J Neurochem 103(6):2491–2504. doi: 10.1111/j.1471-4159.2007.04962.x PubMedCrossRefGoogle Scholar
  44. 44.
    Schober A, Hertel R, Arumae U, Farkas L, Jaszai J, Krieglstein K, Saarma M, Unsicker K (1999) Glial cell line-derived neurotrophic factor rescues target-deprived sympathetic spinal cord neurons but requires transforming growth factor-beta as cofactor in vivo. J Neurosci 19(6):2008–2015PubMedGoogle Scholar
  45. 45.
    Parikh DH, Tam PK, Lloyd DA, Van Velzen D, Edgar DH (1992) Quantitative and qualitative analysis of the extracellular matrix protein, laminin, in Hirschsprung’s disease. J Pediatr Surg 27(8):991–995. pii:0022-3468(92)90545-I (discussion 995–996)Google Scholar
  46. 46.
    Bhattacharyya A, Oppenheim R, Prevette D, Moore B, Brackenbury RNR (1992) S100 is present in developing chicken neurons and Schwann cells and promotes motor neuron survival in vivo. J Neurobiol 23(4):451–466. doi: 10.1002/neu.480230410 PubMedCrossRefGoogle Scholar
  47. 47.
    Ellis EF, Willoughby KA, Sparks SA, Chen T (2007) S100B protein is released from rat neonatal neurons, astrocytes, and microglia by in vitro trauma and anti-S100 increases trauma-induced delayed neuronal injury and negates the protective effect of exogenous S100B on neurons. J Neurochem 101(6):1463–1470. doi: 10.1111/j.1471-4159.2007.04515.x PubMedCrossRefGoogle Scholar
  48. 48.
    Druse MJ, Gillespie RA, Tajuddin NF, Rich M (2007) S100B-mediated protection against the pro-apoptotic effects of ethanol on fetal rhombencephalic neurons. Brain Res 1150:46–64. doi: 10.1016/j.brainres.2007.02.092 PubMedCrossRefGoogle Scholar
  49. 49.
    Sen J, Belli A (2007) S100B in neuropathologic states: The CRP of the brain? J Neurosci Res 85(7):1373–1380. doi: 10.1002/jnr.21211 PubMedCrossRefGoogle Scholar
  50. 50.
    Gazzolo D, Florio P, Zullino E, Giovannini L, Scopesi F, Bellini C, Peri V, Mezzano P, Petraglia F, Michetti F (2010) S100B protein increases in human blood and urine during stressful activity. Clin Chem Lab Med 48(9):1363–1365. doi: 10.1515/CCLM.2010.262 PubMedCrossRefGoogle Scholar
  51. 51.
    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(1):205–216, e206. doi: 10.1053/j.gastro.2008.03.035 Google Scholar
  52. 52.
    Silva AT, Wardhaugh T, Dolatshad NF, Jones S, Saffrey MJ (2008) Neural progenitors from isolated postnatal rat myenteric ganglia: expansion as neurospheres and differentiation in vitro. Brain Res 1218:47–53. doi: 10.1016/j.brainres.2008.04.051 PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Cornelia Irene Hagl
    • 1
  • Sabine Heumüller
    • 1
  • Markus Klotz
    • 2
  • Ulrike Subotic
    • 3
  • Lucas Wessel
    • 1
  • Karl-Herbert Schäfer
    • 1
    • 4
    Email author
  1. 1.Department of Pediatric Surgery, Medical Faculty MannheimUniversity of HeidelbergMannheimGermany
  2. 2.University of Applied SciencesZweibrückenGermany
  3. 3.Department of Pediatric SurgeryUniversity of ZürichZurichSwitzerland
  4. 4.Department of Computer Sciences and Microsystems TechnologyUniversity of Applied Sciences, Life ScienceZweibrückenGermany

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