Journal of Molecular Neuroscience

, Volume 51, Issue 2, pp 307–317 | Cite as

A New Experimental Model for Neuronal and Glial Differentiation Using Stem Cells Derived from Human Exfoliated Deciduous Teeth

  • Akvilė Jarmalavičiūtė
  • Virginijus Tunaitis
  • Eglė Strainienė
  • Rūta Aldonytė
  • Arūnas Ramanavičius
  • Algirdas Venalis
  • Karl-Eric Magnusson
  • Augustas PivoriūnasEmail author


Stem cells isolated from human adult tissues represent a promising source for neural differentiation studies in vitro. We have isolated and characterized stem cells from human exfoliated deciduous teeth (SHEDs). These originate from the neural crest and therefore particularly suitable for induction of neural differentiation. We here established a novel three-stage protocol for neural differentiation of SHEDs cells. After adaptation to a serum-free and neurogenic environment, SHEDs were induced to differentiate. This resulted in the formation of stellate or bipolar round-shaped neuron-like cells with subpopulations expressing markers of sensory neurons (Brn3a, peripherin) and glia (myelin basic protein). Commercial PCR array analyses addressed the expression profiles of genes related to neurogenesis and cAMP/calcium signalling. We found distinct evidence for the upregulation of genes regulating the specification of sensory (MAF), sympathetic (midkine, pleitrophin) and dopaminergic (tyrosine hydroxylase, Nurr1) neurons and the differentiation and support of myelinating and non-myelinating Schwann cells (Krox24, Krox20, apolipoprotein E). Moreover, for genes controlling major developmental signalling pathways, there was upregulation of BMP (TGF β-3, BMP2) and Notch (Notch 2, DLL1, HES1, HEY1, HEY2) in the differentiating SHEDs. SHEDs treated according to our new differentiation protocol gave rise to mixed neuronal/glial cell cultures, which opens new possibilities for in vitro studies of neuronal and glial specification and broadens the potential for the employment of such cells in experimental models and future treatment strategies.


Neural differentiation Glial differentiation Neural crest Mesenchymal stem cells SHED 



This research was supported by a grant (No. MIP-084/2011) from the Research Council of Lithuania and the Swedish Research Council (KEM; No. 2010-3045). We would like to thank Dr. Arūnas Stirkė for technical assistance with the confocal microscopy.

Supplementary material

Supplementary video

After the induction of neural differentiation, SHEDs were monitored continuously in real time for approximately 120 h using Cell-IQ® PC Cell Imaging & Analysis System (CM Technologies) (WMV 23325 kb)

12031_2013_46_MOESM2_ESM.doc (246 kb)
Table S1 Differential expression of genes important for neurogenesis and neural stem cells during neural differentiation of SHEDs. We used RT2 Profiler™ PCR Arrays from SABiosciences, A QIAGEN company (Neurogenesis and Neural Stem Cell array, PAHS-404A). Gene expression levels were analysed using RT2 Profiler PCR Array Data Analysis software (version 3.5, QIAGEN) (DOC 246 kb)
12031_2013_46_MOESM3_ESM.doc (157 kb)
Table S2 Differential expression of genes important for cAMP/Ca2+ signalling during neural differentiation of SHEDs. We used RT2 Profiler™ PCR Arrays from SABiosciences, A QIAGEN company (Human cAMP/Ca2+ PathwayFinder array, PAHS-066A). Gene expression levels were analysed using RT2 Profiler PCR Array Data Analysis software (version 3.5, QIAGEN) (DOC 157 kb)
12031_2013_46_MOESM4_ESM.doc (173 kb)
Fig. S1 Differential expression of genes important for neurogenesis and neural stem cells during neural differentiation of SHEDs. Heatmap was generated using RT2 Profiler PCR Array Data Analysis software (version 3.5, QIAGEN) (DOC 173 kb)
12031_2013_46_MOESM5_ESM.doc (136 kb)
Fig. S2 Differential expression of genes important for cAMP/Ca2+ signalling during neural differentiation of SHEDs. Heatmap was generated using RT2 Profiler PCR Array Data Analysis software (version 3.5, QIAGEN) (DOC 136 kb)
12031_2013_46_MOESM6_ESM.jpg (896 kb)
Fig. S3 Formation of compact multilayer structures similar to the ganglions in differentiating SHEDs (JPEG 895 kb)


  1. Arthur A, Rychkov G, Shi S, Koblar S, Gronthos S (2008) Adult human dental pulp stem cells differentiate toward functionally active neurons under appropriate environmental cues. Stem Cells (Dayton, Ohio) 26(3e615310-8fc0-ce51-d02a-9b75707ef61d):1787–1882. doi: 10.1634/stemcells.2007-0979
  2. Boison D, Chen JF, Fredholm B (2010) Adenosine signaling and function in glial cells. Cell Death Differ 17(d9ebcffd-6d4c-db57-e80c-9b75707e1779):1071–1153. doi: 10.1038/cdd.2009.131
  3. Boyles J, Pitas R, Wilson E, Mahley R, Taylor J (1985) Apolipoprotein E associated with astrocytic glia of the central nervous system and with nonmyelinating glia of the peripheral nervous system. J Clin Investig 76(83a4c912-b23d-bd44-3f66-9b75707e4a2c):1501–1514. doi: 10.1172/jci112130
  4. Clayton K, Podlesniy P, Figueiro-Silva J, López-Doménech G, Benitez L, Enguita M, Abad M, Soriano E, Trullas R (2012) NP1 regulates neuronal activity-dependent accumulation of BAX in mitochondria and mitochondrial dynamics. J Neurosci Off J Soc Neurosci 32(af33b79b-f816-bf11-2b99-9b75708b1837):1453–1519. doi: 10.1523/jneurosci.4604-11.2012
  5. Cox ME, Deeble PD, Lakhani S, Parsons SJ (1999) Acquisition of neuroendocrine characteristics by prostate tumor cells is reversible. Cancer Res (0a29f16c-c722-058b-ff7f-9b75707efea5)Google Scholar
  6. da Silva Meirelles L, Chagastelles PC, Nardi NB (2006) Mesenchymal stem cells reside in virtually all post-natal organs and tissues. J Cell Sci 119(Pt 11):2204–2213PubMedCrossRefGoogle Scholar
  7. da Silva Meirelles L, Caplan AI, Nardi NB (2008) In search of the in vivo identity of mesenchymal stem cells. Stem Cells 26(9):2287–2299. doi: 10.1634/stemcells.2007-1122 PubMedCrossRefGoogle Scholar
  8. Falk A, Frisen J (2002) Amphiregulin is a mitogen for adult neural stem cells. J Neurosci Res 69(6):757–762. doi: 10.1002/jnr.10410 PubMedCrossRefGoogle Scholar
  9. Fernandes K, McKenzie I, Mill P, Smith K, Akhavan M, Barnabé-Heider F, Biernaskie J, Junek A, Kobayashi N, Toma J, Kaplan D, Labosky P, Rafuse V, Hui C-C, Miller F (2004) A dermal niche for multipotent adult skin-derived precursor cells. Nat Cell Biol 6(bce5f16a-0182-7b99-10cd-9b75707d6d33):1082–1175. doi: 10.1038/ncb1181
  10. Gögel S, Gubernator M, Minger S (2011) Progress and prospects: stem cells and neurological diseases. Gene Ther 18(e4417058-6d0c-3c9f-100a-9b75708f5453):1–7. doi: 10.1038/gt.2010.130
  11. Goulburn AL, Stanley EG, Elefanty AG, Anderson SA (2012) Generating GABAergic cerebral cortical interneurons from mouse and human embryonic stem cells. Stem Cell Res 8(3):416–426. doi: 10.1016/j.scr.2011.12.002 PubMedCrossRefGoogle Scholar
  12. Gregory CA, Reyes E, Whitney MJ, Spees JL (2006) Enhanced engraftment of mesenchymal stem cells in a cutaneous wound model by culture in allogenic species-specific serum and administration in fibrin constructs. Stem Cells 24(10):2232–2243. doi: 10.1634/stemcells.2005-0612 PubMedCrossRefGoogle Scholar
  13. Helfand B, Loomis P, Yoon M, Goldman R (2003) Rapid transport of neural intermediate filament protein. J Cell Sci 116(8fc58920-c9e7-0298-6079-9b75708bb2e5):2345–2404. doi: 10.1242/jcs.00526
  14. Henley S, Dick F (2012) The retinoblastoma family of proteins and their regulatory functions in the mammalian cell division cycle. Cell Div 7(80ebeaa3-3d3f-3dd6-125e-9b75707fb83b):10. doi: 10.1186/1747-1028-7-10
  15. Hu B-Y, Zhang S-C (2009) Differentiation of spinal motor neurons from pluripotent human stem cells. Nat Protoc 4(01a5a69c-6b08-f653-a530-9b75708101f2):1295–1599. doi: 10.1038/nprot.2009.127
  16. Hu BY, Du ZW, Li XJ, Ayala M, Zhang SC (2009) Human oligodendrocytes from embryonic stem cells: conserved SHH signaling networks and divergent FGF effects. Development 136(9):1443–1452. doi: 10.1242/dev.029447 PubMedCrossRefGoogle Scholar
  17. Jankovic J, Chen S, Le W (2005) The role of Nurr1 in the development of dopaminergic neurons and Parkinson's disease. Prog Neurobiol 77(920926b6-02b5-cf78-8839-9b757090d425):128–166. doi: 10.1016/j.pneurobio.2005.09.001
  18. Kenzelmann D, Chiquet-Ehrismann R, Tucker R (2007) Teneurins, a transmembrane protein family involved in cell communication during neuronal development. Cell Mol Life Sci CMLS 64(70fb9120-2d40-b40b-2e8e-9b75708f4ef3):1452–1458. doi: 10.1007/s00018-007-7108-9
  19. Kim SU, de Vellis J (2009) Stem cell-based cell therapy in neurological diseases: a review. J Neurosci Res 87(10):2183–2200. doi: 10.1002/jnr.22054 PubMedCrossRefGoogle Scholar
  20. Király M, Porcsalmy B, Pataki A, Kádár K, Jelitai M, Molnár B, Hermann P, Gera I, Grimm W-D, Ganss B, Zsembery A, Varga G (2009) Simultaneous PKC and cAMP activation induces differentiation of human dental pulp stem cells into functionally active neurons. Neurochem Int 55(b485ca2b-ca3f-694a-1095-9b75707c3214):323–355. doi: 10.1016/j.neuint.2009.03.017
  21. Lee G, Kim H, Elkabetz Y, Al Shamy G, Panagiotakos G, Barberi T, Tabar V, Studer L (2007) Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells. Nat Biotechnol. 25(f29653c4-2875-7230-5e45-9b757081335a):1468–1543. doi: 10.1038/nbt1365
  22. Li L, Hung A, Porter A (2008) Secretogranin II: a key AP-1-regulated protein that mediates neuronal differentiation and protection from nitric oxide-induced apoptosis of neuroblastoma cells. Cell Death Differ 15 (6318b9d1-0b05-081d-bbd1-9b75708cbe46):879–967. doi: 10.1038/cdd.2008.8
  23. Lindvall O, Barker RA, Brüstle O, Isacson O, Svendsen CN (2012) Clinical translation of stem cells in neurodegenerative disorders. Cell Stem Cell 10(2):151–155PubMedCrossRefGoogle Scholar
  24. Massague J (2012) TGFbeta signalling in context. Nat Rev Mol Cell Biol 13(10):616–630. doi: 10.1038/nrm3434 PubMedCrossRefGoogle Scholar
  25. Morrison S, Perez S, Qiao Z, Verdi J, Hicks C, Weinmaster G, Anderson D (2000) Transient Notch activation initiates an irreversible switch from neurogenesis to gliogenesis by neural crest stem cells. Cell 101(2d4b829c-0aab-3b20-2934-9b757091b123):499–1009Google Scholar
  26. Pavan W, Raible D (2012) Specification of neural crest into sensory neuron and melanocyte lineages. Dev Biol 366(0b304e3b-7e59-a81d-af3d-9b75708e26b3):55–118. doi: 10.1016/j.ydbio.2012.02.038
  27. Pfisterer U, Kirkeby A, Torper O, Wood J, Nelander J, Dufour A, Bjorklund A, Lindvall O, Jakobsson J, Parmar M (2011) Direct conversion of human fibroblasts to dopaminergic neurons. Proc Natl Acad Sci U S A 108(25):10343–10348. doi: 10.1073/pnas.1105135108 PubMedCrossRefGoogle Scholar
  28. Pintér E, Helyes Z, Szolcsányi J (2006) Inhibitory effect of somatostatin on inflammation and nociception. Pharmacol Ther 112(70eb4bec-86c1-3a8d-9dd5-9b75708541e8):440–496. doi: 10.1016/j.pharmthera.2006.04.010
  29. Pivoriuunas A, Surovas A, Borutinskaite V, Matuzeviccius D, Treigyte G, Savickiene J, Tunaitis V, Aldonyte R, Jarmalavicciuute A, Suriakaite K, Liutkeviccius E, Venalis A, Navakauskas D, Navakauskiene R, Magnusson KE (2010) Proteomic analysis of stromal cells derived from the dental pulp of human exfoliated deciduous teeth. Stem Cells Dev 19(7):1081–1093. doi: 10.1089/scd.2009.0315 PubMedCrossRefGoogle Scholar
  30. Reiff T, Huber L, Kramer M, Delattre O, Janoueix-Lerosey I, Rohrer H (2011) Midkine and Alk signaling in sympathetic neuron proliferation and neuroblastoma predisposition. Dev (Cambridge, England) 138(83d1ac7c-61bf-ff03-f90e-9b75707ea540):4699–5407. doi: 10.1242/dev.072157
  31. Robinton DA, Daley GQ (2012) The promise of induced pluripotent stem cells in research and therapy. Nature 481(7381):295–305. doi: 10.1038/nature10761 PubMedCrossRefGoogle Scholar
  32. Santagati F, Rijli F (2003) Cranial neural crest and the building of the vertebrate head. Nat Rev Neurosci 4(b6514d1d-a1db-b4f3-d776-9b757080eeab):806–824. doi: 10.1038/nrn1221
  33. Takashima Y, Era T, Nakao K, Kondo S, Kasuga M, Smith A, Nishikawa S-I (2007) Neuroepithelial cells supply an initial transient wave of MSC differentiation. Cell 129(3f2ac373-b298-8035-5cbc-9b757089eb0e):1377–1465. doi: 10.1016/j.cell.2007.04.028
  34. Thier M, Worsdorfer P, Lakes YB, Gorris R, Herms S, Opitz T, Seiferling D, Quandel T, Hoffmann P, Nothen MM, Brustle O, Edenhofer F (2012) Direct conversion of fibroblasts into stably expandable neural stem cells. Cell Stem Cell 10(4):473–479. doi: 10.1016/j.stem.2012.03.003 PubMedCrossRefGoogle Scholar
  35. Topilko P, Levi G, Merlo G, Mantero S, Desmarquet C, Mancardi G, Charnay P (1997) Differential regulation of the zinc finger genes Krox-20 and Krox-24 (Egr-1) suggests antagonistic roles in Schwann cells. J Neurosci Res 50(8993b146-941b-52fa-b687-9b757081bdf0):702–714Google Scholar
  36. Vierbuchen T, Ostermeier A, Pang ZP, Kokubu Y, Sudhof TC, Wernig M (2010) Direct conversion of fibroblasts to functional neurons by defined factors. Nature 463(7284):1035–1041. doi: 10.1038/nature08797 PubMedCrossRefGoogle Scholar
  37. Vodrazka P, Korostylev A, Hirschberg A, Swiercz JM, Worzfeld T, Deng S, Fazzari P, Tamagnone L, Offermanns S, Kuner R (2009) The semaphorin 4D-plexin-B signalling complex regulates dendritic and axonal complexity in developing neurons via diverse pathways. Eur J Neurosci 30(7):1193–1208. doi: 10.1111/j.1460-9568.2009.06934.x PubMedCrossRefGoogle Scholar
  38. Vodyanik MA, Yu J, Zhang X, Tian S, Stewart R, Thomson JA, Slukvin II (2010) A mesoderm-derived precursor for mesenchymal stem and endothelial cells. Cell Stem Cell 7(6):718–729. doi: 10.1016/j.stem.2010.11.011 PubMedCrossRefGoogle Scholar
  39. Wang J, Wang X, Sun Z, Wang X, Yang H, Shi S, Wang S (2010) Stem cells from human-exfoliated deciduous teeth can differentiate into dopaminergic neuron-like cells. Stem Cells Dev 19(938c6087-b406-d3e0-b89e-9b75708260cb):1375–1458. doi: 10.1089/scd.2009.0258
  40. Wang T, Xiong J-Q, Ren X-B, Sun W (2012) The role of Nogo-A in neuroregeneration: a review. Brain Res Bull 87(c7074f99-df20-b59c-87fd-9b757090125b):499–1002. doi: 10.1016/j.brainresbull.2012.02.011
  41. Wende H, Lechner S, Cheret C, Bourane S, Kolanczyk M, Pattyn A, Reuter K, Munier F, Carroll P, Lewin G, Birchmeier C (2012) The transcription factor c-Maf controls touch receptor development and function. Science (New York, NY) 335 (4d54389e-5a5d-243b-9c18-9b7570901825):1373–1379. doi: 10.1126/science.1214314
  42. Wu SM, Hochedlinger K (2011) Harnessing the potential of induced pluripotent stem cells for regenerative medicine. Nat Cell Biol 13(5):497–505PubMedCrossRefGoogle Scholar
  43. Yan Y, Yang D, Zarnowska ED, Du Z, Werbel B, Valliere C, Pearce RA, Thomson JA, Zhang SC (2005) Directed differentiation of dopaminergic neuronal subtypes from human embryonic stem cells. Stem Cells 23(6):781–790. doi: 10.1634/stemcells.2004-0365 PubMedCrossRefGoogle Scholar
  44. Yin C, Zhou S, Jiang L, Sun X (2012) Mechanical injured neurons stimulate astrocytes to express apolipoprotein E through ERK pathway. Neurosci Lett 515(d9063f17-c03a-0902-95f6-9b757086b5d9):77–158. doi: 10.1016/j.neulet.2012.03.023
  45. Zhang SC, Wernig M, Duncan ID, Brustle O, Thomson JA (2001) In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat Biotechnol 19(12):1129–1133. doi: 10.1038/nbt1201-1129 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Akvilė Jarmalavičiūtė
    • 1
    • 2
  • Virginijus Tunaitis
    • 1
  • Eglė Strainienė
    • 3
  • Rūta Aldonytė
    • 1
  • Arūnas Ramanavičius
    • 4
  • Algirdas Venalis
    • 1
  • Karl-Eric Magnusson
    • 5
  • Augustas Pivoriūnas
    • 1
    Email author
  1. 1.Department of Stem Cell BiologyState Research Institute Centre for Innovative MedicineVilniusLithuania
  2. 2.Department of Neurobiology and Biophysics, Faculty of Natural SciencesVilnius UniversityVilniusLithuania
  3. 3.Department of Chemistry and BioengineeringVilnius Gediminas Technical UniversityVilniusLithuania
  4. 4.Department of Physical Chemistry, Faculty of ChemistryVilnius UniversityVilniusLithuania
  5. 5.Department of Clinical and Experimental MedicineLinköping UniversityLinköpingSweden

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