Differentiation of GDNF and NT-3 dual gene-modified rat bone marrow mesenchymal stem cells into enteric neuron-like cells

Abstract

Bone marrow mesenchymal stem cells (BMSCs) have been shown to be multipotent cells that possess high self-replicating capacity. The purpose of our study was to investigate the feasibility of using enteric neuron-like cells obtained by in vitro induction and differentiated from rat BMSCs for the treatment of Hirschsprung’s disease (HD). Glial cell-derived neurotrophic factor (GDNF) and neurotrophin-3 (NT-3) are neurotrophic factors that play important roles in neuronal development, differentiation, survival and function. Meanwhile, GDNF mutations are a major cause of HD. In this study, BMSCs were transfected with eukaryotic expression plasmids co-expressing GDNF and NT-3, and the transfected cells displayed neuron-like changes after differentiation induced by fetal gut culture medium (FGCM). Immunofluorescence assay showed positive expression of the neuronal marker NSE and the enteric neuronal markers PGP9.5, VIP and nNOS. Reverse transcription-polymerase chain reaction (RT-PCR) revealed the expression of GDNF and NT-3 in transfected BMSCs. The present study indicates that genetically modified BMSCs co-expressing GDNF and NT-3 are able to differentiate into enteric neuronal cells and express enteric nerve markers when induced by FGCM. This study provides an experimental basis for gene therapy to treat enteric nervous system-related disorders, such as HD.

Keywords

mesenchymal stem cell / glial cell-derived neurotrophic factor / neurotrophin-3 / enteric neurons / cell transfection

References

[1] KimH.Y., OhJ.T.. Stabilization period after 1-stage transanal endorectal pull-through operation for Hirschsprung disease. J Pediatr Surg, 2009, 44(9): 1799-1804

[2] TannuriA.C., TannuriU., RomãoR.L.. Transanal endorectal pull-through in children with Hirschsprung’s disease — technical refinements and comparison of results with the Duhamel procedure. J Pediatr Surg, 2009, 44(4): 767-772

[3] NatarajanD., Marcos-GutierrezC., PachnisV., et al.. Requirement of signalling by receptor tyrosine kinase RET for the directed migration of enteric nervous system progenitor cells during mammalian embryogenesis. Development, 2002, 129(22): 5151-5160

[4] KennyS.E., TamP.K., Garcia-BarceloM.. Hirschsprung’s disease. Semin Pediatr Surg, 2010, 19(3): 194-200

[5] DelormeB., ChateauvieuxS., CharbordP.. The concept of mesenchymal stem cells. Regen Med, 2006, 1(4): 497-509

[6] LiL., LiF., QiH., et al.. Coexpression of Pdx1 and betacellulin in mesenchymal stem cells could promote the differentiation of nestin-positive epithelium-like progenitors and pancreatic islet-like spheroids. Stem Cells Dev, 2008, 17(4): 815-823

[7] BosnakovskiD., MizunoM., KimG., et al.. Isolation and multilineage differentiation of bovine bone marrow mesenchymal stem cells. Cell Tissue Res, 2005, 319(2): 243-253

[8] KodaM., OkadaS., NakayamaT., et al.. Hematopoietic stem cell and marrow stromal cell for spinal cord injury in mice. Neuroreport, 2005, 16(16): 1763-1767

[9] KamadaT., KodaM., DezawaM., et al.. Transplantation of bone marrow stromal cell-derived Schwann cells promotes axonal regeneration and functional recovery after complete transaction of adult rat spinal cord. J Neuropathol Exp Neurol, 2005, 64(1): 37-45

[10] MetzgerM.. Neurogenesis in the enteric nervous system. Arch Ital Biol, 2010, 148(2): 73-83

[11] WangH., HughesI., PlanerW., et al.. The timing and location of glial cell line-derived neurotrophic factor expression determine enteric nervous system structure and function. J Neurosci, 2010, 30(4): 1523-1538

[12] FernandezR.M., Ruiz-FerrerM., Lopez-AlonsoM., et al.. Polymorphisms in the genes encoding the 4 RET ligands, GDNF, NTN, ARTN, PSPN, and susceptibility to Hirschsprung disease. J Pediatr Surg, 2008, 43(11): 2042-2047

[13] ZhangY., PardridgeW.M.. Near complete rescue of experimental Parkinson’s disease with intravenous, nonviral GDNF gene therapy. Pharm Res, 2009, 26(5): 1059-1063

[14] FoxE.A., McAdamsJ.. Smooth-muscle-specific expression of neurotrophin-3 in mouse embryonic and neonatal gastrointestinal tract. Cell Tissue Res, 2010, 340(2): 267-286

[15] SahenkZ., GallowayG., EdwardsC., et al.. TrkB and TrkC agonist antibodies improve function, electrophysiologic and pathologic features in Trembler J mice. Exp Neurol, 2010, 224(2): 495-506

[16] TropelP., PlatetN., PlatelJ.C., et al.. Functional neuronal differentiation of bone marrow-derived mesenchymal stem cells. Stem Cells, 2006, 24(12): 2868-2876

[17] ParkJ.S., HashiC., LiS.. Culture of bone marrow mesenchymal stem cells on engineered matrix. Methods Mol Biol, 2010, 621: 117-137

[18] YangH.M., ZhangS.M., ZhengR.Y.. Whether the ion channel of differentiation from bone marrow mesenchymal stem cells intoneural-like cells has electrophysiological characteristics?. J Clin Rehab Tissue Eng Res, 2009, 13(40): 7881-7884