Advertisement

Pharmacological Notch pathway inhibition leads to cell cycle arrest and stimulates ascl1 and neurogenin2 genes expression in dental pulp stem cells-derived neurospheres

  • Ali NiapourEmail author
  • Hatef Ghasemi Hamidabadi
  • Nazila Niapour
  • Perham Mohammadi
  • Marzieh Sharifi Pasandi
  • Vadoud Malekzadeh
Original Research Paper
  • 38 Downloads

Abstract

Objective

Human dental pulp-derived stem cells (hDPSCs) are becoming an attractive source for cell-based neurorestorative therapies. As such, it is important to understand the molecular mechanisms that regulate the differentiation of hDPSCs toward the neuronal fate. Notch signaling plays key roles in neural stem/progenitor cells (NS/PCs) maintenance and prevention of their differentiation. The aim of this study was to address the effects of Notch signaling inhibition on neurosphere formation of hDPSCs and neuronal differentiation of hDPSCs-neurospheres.

Results

hDPSCs were isolated from third molar teeth. The cultivated hDPSCs highly expressed CD90 and CD44 and minimally presented CD34 and CD45 surface markers. The osteo/adipogenic differentiation of hDPSCs was documented. hDPSCs were cultured in neural induction medium and N-[N-(3,5-difluorophenacetyl-l-alanyl)]-Sphenylglycine t-butyl ester (DAPT) was applied to impede Notch signaling during transformation into spheres or on the formed neurospheres. Our results showed that the size and number of neurospheres decreased and the expression profile of nestin, sox1 and pax6 genes reduced provided DAPT. Treatment of the formed neurospheres with DAPT resulted in the cleaved Notch1 reduction, G0/G1 arrest and a decline in L-lactate production. DAPT significantly reduced hes1 and hey1 genes, while ascl1 and neurogenin2 expressions augmented. The number of MAP2 positive cells improved in the DAPT-treated group.

Conclusions

Our findings demonstrated the Notch activity in hDPSCs-neurospheres. DAPT treatment positively regulated proneural genes expression and increased neuronal-like differentiation.

Keywords

hDPSCs Notch signaling Gamma-secretase inhibitor Neurosphere Differentiation ascl1 Neurogenin2 

Notes

Acknowledgements

This study was financially supported by a Research Project (Grant No. 9223) of Research Vice-Chancellery of Ardabil University of Medical Sciences, Iran.

Author contributions

hDPSCs isolation and culture, neural induction, MTT assay and cell cycle were done by AN. hDPSCs characterization was done by VM. Real time-PCR was performed and interpreted by AN and NN. Western blotting and immunofluorescent staining were performed by PM and HGH, respectively. The l-lactate assay was performed by MSP. The paper was written principally by AN with input from all the other authors especially PM. Study design and statistically analyzing of data were done by AN and HGH.

Compliance with ethical standards

Conflict of interest

The authors declared no potential conflicts of interest.

Supplementary material

10529_2019_2687_MOESM1_ESM.rar (114 kb)
Supplementary material 1 (RAR 114 kb)
10529_2019_2687_MOESM2_ESM.rar (301 kb)
Supplementary material 2 (RAR 300 kb)
10529_2019_2687_MOESM3_ESM.rar (47 kb)
Supplementary material 3 (RAR 47 kb)

References

  1. Abe S, Hamada K, Miura M, Yamaguchi S (2012) Neural crest stem cell property of apical pulp cells derived from human developing tooth. Cell Biol Int 36:927–936CrossRefGoogle Scholar
  2. Ali F, Hindley C, McDowell G, Deibler R, Jones A, Kirschner M et al (2011) Cell cycle-regulated multi-site phosphorylation of Neurogenin 2 coordinates cell cycling with differentiation during neurogenesis. Development 138:4267–4277CrossRefGoogle Scholar
  3. Ali FR, Cheng K, Kirwan P, Metcalfe S, Livesey FJ, Barker RA, Philpott A (2014) The phosphorylation status of Ascl1 is a key determinant of neuronal differentiation and maturation in vivo and in vitro. Development 141:2216–2224CrossRefGoogle Scholar
  4. Arthur A, Rychkov G, Shi S, Koblar SA, Gronthos S (2008) Adult human dental pulp stem cells differentiate toward functionally active neurons under appropriate environmental cues. Stem Cells 26:1787–1795CrossRefGoogle Scholar
  5. Azari H, Reynolds BA (2016) In vitro models for neurogenesis. Cold Spring Harb Perspect Biol 8:a021279CrossRefGoogle Scholar
  6. Ben-Shushan E, Feldman E, Reubinoff BE (2015) Notch signaling regulates motor neuron differentiation of human embryonic stem cells. Stem Cells 33:403–415CrossRefGoogle Scholar
  7. Bi P, Kuang S (2015) Notch signaling as a novel regulator of metabolism. Trends Endocrinol Metab 26:248–255CrossRefGoogle Scholar
  8. Borghese L, Dolezalova D, Opitz T, Haupt S, Leinhaas A, Steinfarz B et al (2010) Inhibition of notch signaling in human embryonic stem cell-derived neural stem cells delays G1/S phase transition and accelerates neuronal differentiation in vitro and in vivo. Stem Cells 28:955–964CrossRefGoogle Scholar
  9. Braune EB, Lendahl U (2016) Notch—a goldilocks signaling pathway in disease and cancer therapy. Discov Med 21:189–196Google Scholar
  10. Bray SJ (2016) Notch signalling in context. Nat Rev Mol Cell Biol 17:722–735CrossRefGoogle Scholar
  11. Chung CS, Fujita N, Kawahara N, Yui S, Nam E, Nishimura R (2013) A comparison of neurosphere differentiation potential of canine bone marrow-derived mesenchymal stem cells and adipose-derived mesenchymal stem cells. J Vet Med Sci 75:879–886CrossRefGoogle Scholar
  12. Dennis DJ, Han S, Schuurmans C (2018) bHLH transcription factors in neural development, disease, and reprogramming. Brain Res 1705:48–65CrossRefGoogle Scholar
  13. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D et al (2006) Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8:315–317CrossRefGoogle Scholar
  14. Gervois P, Struys T, Hilkens P, Bronckaers A, Ratajczak J, Politis C et al (2015) Neurogenic maturation of human dental pulp stem cells following neurosphere generation induces morphological and electrophysiological characteristics of functional neurons. Stem Cells Dev 24:296–311CrossRefGoogle Scholar
  15. Goorha S, Reiter LT (2017) Culturing and neuronal differentiation of human dental pulp stem cells. Curr Protoc Hum Genet 92(1):21–26Google Scholar
  16. Gronthos S, Arthur A, Bartold PM, Shi S (2011) A method to isolate and culture expand human dental pulp stem cells. Methods Mol Biol 698:107–121CrossRefGoogle Scholar
  17. Gu W, Gaeta X, Sahakyan A, Chan AB, Hong CS, Kim R et al (2016) Glycolytic metabolism plays a functional role in regulating human pluripotent stem cell state. Cell Stem Cell 19:476–490CrossRefGoogle Scholar
  18. Hansson ML, Behmer S, Ceder R, Mohammadi S, Preta G, Grafstrom RC et al (2012) MAML1 acts cooperatively with EGR1 to activate EGR1-regulated promoters: implications for nephrogenesis and the development of renal cancer. PLoS ONE 7:e46001CrossRefGoogle Scholar
  19. Heng BC, Lim LW, Wu W, Zhang C (2016) An overview of protocols for the neural induction of dental and oral stem cells in vitro. Tissue Eng Part B 22:220–250CrossRefGoogle Scholar
  20. Hollands P, Aboyeji D, Orcharton M (2018) Dental pulp stem cells in regenerative medicine. Br Dent JGoogle Scholar
  21. Imayoshi I, Sakamoto M, Yamaguchi M, Mori K, Kageyama R (2010) Essential roles of Notch signaling in maintenance of neural stem cells in developing and adult brains. J Neurosci 30:3489–3498CrossRefGoogle Scholar
  22. Kadar K, Kiraly M, Porcsalmy B, Molnar B, Racz GZ, Blazsek J et al (2009) Differentiation potential of stem cells from human dental origin—promise for tissue engineering. J Physiol Pharmacol 60(Suppl 7):167–175Google Scholar
  23. Kageyama R, Ohtsuka T, Hatakeyama J, Ohsawa R (2005) Roles of bHLH genes in neural stem cell differentiation. Exp Cell Res 306:343–348CrossRefGoogle Scholar
  24. Kaukua N, Shahidi MK, Konstantinidou C, Dyachuk V, Kaucka M, Furlan A et al (2014) Glial origin of mesenchymal stem cells in a tooth model system. Nature 513:551–554CrossRefGoogle Scholar
  25. Lin HT, Otsu M, Nakauchi H (2013) Stem cell therapy: an exercise in patience and prudence. Philos Trans R Soc Lond B 368:20110334CrossRefGoogle Scholar
  26. Lindvall O, Barker RA, Brustle O, Isacson O, Svendsen CN (2012) Clinical translation of stem cells in neurodegenerative disorders. Cell Stem Cell 10:151–155CrossRefGoogle Scholar
  27. Liu Y, Li P, Liu K, He Q, Han S, Sun X et al (2014) Timely inhibition of Notch signaling by DAPT promotes cardiac differentiation of murine pluripotent stem cells. PLoS ONE 9:e109588CrossRefGoogle Scholar
  28. Lleo A, Saura CA (2011) gamma-secretase substrates and their implications for drug development in Alzheimer’s disease. Curr Top Med Chem 11:1513–1527CrossRefGoogle Scholar
  29. Lowell S, Benchoua A, Heavey B, Smith AG (2006) Notch promotes neural lineage entry by pluripotent embryonic stem cells. PLoS Biol 4:e121CrossRefGoogle Scholar
  30. Luo L, He Y, Wang X, Key B, Lee BH, Li H, Ye Q (2018) Potential roles of dental pulp stem cells in neural regeneration and repair. Stem Cells Int 2018:1731289Google Scholar
  31. Ma K, Fox L, Shi G, Shen J, Liu Q, Pappas JD et al (2011) Generation of neural stem cell-like cells from bone marrow-derived human mesenchymal stem cells. Neurol Res 33:1083–1093CrossRefGoogle Scholar
  32. Mortada I, Mortada R, Al Bazzal M (2018) Dental pulp stem cells and the management of neurological diseases: an update. J Neurosci Res 96:265–272CrossRefGoogle Scholar
  33. Mukai T, Nagamura-Inoue T, Shimazu T, Mori Y, Takahashi A, Tsunoda H et al (2016) Neurosphere formation enhances the neurogenic differentiation potential and migratory ability of umbilical cord-mesenchymal stromal cells. Cytotherapy 18:229–241CrossRefGoogle Scholar
  34. Mussmann C, Hubner R, Trilck M, Rolfs A, Frech MJ (2014) HES5 is a key mediator of Wnt-3a-induced neuronal differentiation. Stem Cells Dev 23:1328–1339CrossRefGoogle Scholar
  35. Niapour N, Tagipour Z, Salehi H, Bagheri A, Rohani A, Talebi M et al (2015) Isolation and identification of mesenchymal and neural crest characteristics of dental pulp derived stem cells. Koomesh 16:520–526Google Scholar
  36. Ohtsuka T, Ishibashi M, Gradwohl G, Nakanishi S, Guillemot F, Kageyama R (1999) Hes1 and Hes5 as notch effectors in mammalian neuronal differentiation. EMBO J 18:2196–2207CrossRefGoogle Scholar
  37. Osathanon T, Manokawinchoke J, Nowwarote N, Aguilar P, Palaga T, Pavasant P (2013) Notch signaling is involved in neurogenic commitment of human periodontal ligament-derived mesenchymal stem cells. Stem Cells Dev 22:1220–1231CrossRefGoogle Scholar
  38. Park NI, Guilhamon P, Desai K, McAdam RF, Langille E, O’Connor M et al (2017) ASCL1 reorganizes chromatin to direct neuronal fate and suppress tumorigenicity of glioblastoma stem cells. Cell Stem Cell 21(209–224):e207Google Scholar
  39. Patrad E, Niapour A, Farassati F, Amani M (2018) Combination treatment of all-trans retinoic acid (ATRA) and gamma-secretase inhibitor (DAPT) cause growth inhibition and apoptosis induction in the human gastric cancer cell line. Cytotechnology 70:865–877CrossRefGoogle Scholar
  40. Pierfelice T, Alberi L, Gaiano N (2011) Notch in the vertebrate nervous system: an old dog with new tricks. Neuron 69:840–855CrossRefGoogle Scholar
  41. Pisciotta A, Bertoni L, Riccio M, Mapelli J, Bigiani A, La Noce M et al (2018) Use of a 3D floating sphere culture system to maintain the neural crest-related properties of human dental pulp stem cells. Front Physiol 9:547CrossRefGoogle Scholar
  42. Razavi S, Mostafavi FS, Mardani M, Zarkesh Esfahani H, Kazemi M, Esfandiari E (2014) Effect of T3 hormone on neural differentiation of human adipose derived stem cells. Cell Biochem Funct 32:702–710CrossRefGoogle Scholar
  43. Rietze RL, Reynolds BA (2006) Neural stem cell isolation and characterization. Methods Enzymol 419:3–23CrossRefGoogle Scholar
  44. Rodas-Junco BA, Villicana C (2017) Dental pulp stem cells: current advances in isolation, expansion and preservation. Tissue Eng Regen Med 14:333–347CrossRefGoogle Scholar
  45. Rosemann A (2015) Stem cell treatments for neurodegenerative diseases: challenges from a science, business and healthcare perspective. Neurodegener Dis Manag 5:85–87CrossRefGoogle Scholar
  46. Salehi H, Amirpour N, Niapour A, Razavi S (2016) An overview of neural differentiation potential of human adipose derived stem cells. Stem Cell Rev 12:26–41CrossRefGoogle Scholar
  47. Sarmento LM, Huang H, Limon A, Gordon W, Fernandes J, Tavares MJ et al (2005) Notch1 modulates timing of G1-S progression by inducing SKP2 transcription and p27 Kip1 degradation. J Exp Med 202:157–168CrossRefGoogle Scholar
  48. Schlett K, Czirok A, Tarnok K, Vicsek T, Madarasz E (2000) Dynamics of cell aggregation during in vitro neurogenesis by immortalized neuroectodermal progenitors. J Neurosci Res 60:184–194CrossRefGoogle Scholar
  49. Sharifi Pasandi M, Hosseini Shirazi F, Gholami MR, Salehi H, Najafzadeh N, Mazani M et al (2017) Epi/perineural and Schwann cells as well as perineural sheath integrity are affected following 2,4-D exposure. Neurotox Res 32:624–638CrossRefGoogle Scholar
  50. Sierra-Sanchez A, Ordonez-Luque A, Espinosa-Ibanez O, Ruiz-Garcia A, Arias-Santiago S (2018) Epithelial in vitro differentiation of mesenchymal stem cells. Curr Stem Cell Res Ther 13:409–422CrossRefGoogle Scholar
  51. Stevens A, Zuliani T, Olejnik C, LeRoy H, Obriot H, Kerr-Conte J et al (2008) Human dental pulp stem cells differentiate into neural crest-derived melanocytes and have label-retaining and sphere-forming abilities. Stem Cells Dev 17:1175–1184CrossRefGoogle Scholar
  52. Stocchetti N, Zanier ER (2016) Chronic impact of traumatic brain injury on outcome and quality of life: a narrative review. Crit Care 20:148CrossRefGoogle Scholar
  53. Tarnok K, Pataki A, Kovacs J, Schlett K, Madarasz E (2002) Stage-dependent effects of cell-to-cell connections on in vitro induced neurogenesis. Eur J Cell Biol 81:403–412CrossRefGoogle Scholar
  54. Thomas JL, Baker K, Han J, Calvo C, Nurmi H, Eichmann AC, Alitalo K (2013) Interactions between VEGFR and Notch signaling pathways in endothelial and neural cells. Cell Mol Life Sci 70:1779–1792CrossRefGoogle Scholar
  55. Wang L, Cheng L, Wang H, Pan H, Yang H, Shao M, Hu T (2016) Glycometabolic reprogramming associated with the initiation of human dental pulp stem cell differentiation. Cell Biol Int 40:308–317CrossRefGoogle Scholar
  56. Ware M, Hamdi-Roze H, Le Friec J, David V, Dupe V (2016) Regulation of downstream neuronal genes by proneural transcription factors during initial neurogenesis in the vertebrate brain. Neural Dev 11:22CrossRefGoogle Scholar
  57. Wilkinson G, Dennis D, Schuurmans C (2013) Proneural genes in neocortical development. Neuroscience 253:256–273CrossRefGoogle Scholar
  58. Wolfe MS (2009) Intramembrane-cleaving proteases. J Biol Chem 284:13969–13973CrossRefGoogle Scholar
  59. Woo SM, Kim J, Han HW, Chae JI, Son MY, Cho S et al (2009) Notch signaling is required for maintaining stem-cell features of neuroprogenitor cells derived from human embryonic stem cells. BMC Neurosci 10:97CrossRefGoogle Scholar
  60. Wu SM, Tan KS, Chen H, Beh TT, Yeo HC, Ng SK et al (2012) Enhanced production of neuroprogenitors, dopaminergic neurons, and identification of target genes by overexpression of sonic hedgehog in human embryonic stem cells. Stem Cells Dev 21:729–741CrossRefGoogle Scholar
  61. Yoon K, Gaiano N (2005) Notch signaling in the mammalian central nervous system: insights from mouse mutants. Nat Neurosci 8:709–715CrossRefGoogle Scholar
  62. Zhang C, Chang J, Sonoyama W, Shi S, Wang CY (2008) Inhibition of human dental pulp stem cell differentiation by Notch signaling. J Dent Res 87:250–255CrossRefGoogle Scholar
  63. Zhang R, Engler A, Taylor V (2018) Notch: an interactive player in neurogenesis and disease. Cell Tissue Res 371:73–89CrossRefGoogle Scholar
  64. Zheng X, Boyer L, Jin M, Mertens J, Kim Y, Ma L et al (2016) Metabolic reprogramming during neuronal differentiation from aerobic glycolysis to neuronal oxidative phosphorylation. Elife 5:e13374CrossRefGoogle Scholar
  65. Zhou ZD, Kumari U, Xiao ZC, Tan EK (2010) Notch as a molecular switch in neural stem cells. IUBMB Life 62:618–623CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Research Laboratory for Embryology and Stem Cells, Department of Anatomical Sciences, School of MedicineArdabil University of Medical SciencesArdabilIran
  2. 2.Department of Anatomy and Cell Biology, Immunogenetic Research Center, Faculty of MedicineMazandaran University of Medical SciencesSariIran
  3. 3.Department of Anatomy & Cell Biology, Faculty of MedicineMazandaran University of Medical SciencesSariIran
  4. 4.Department of Pharmacology and Toxicology, School of PharmacyArdabil University of Medical SciencesArdabilIran
  5. 5.Molecular and Cell Biology Research Center, Faculty of MedicineMazandaran University of Medical SciencesSariIran

Personalised recommendations