MicroRNA Expression Profiling by PCR Array in 2D and 3D Differentiated Neural Culture Systems and Target Validation

  • Lara StevanatoEmail author
  • Caroline Hicks
  • Lavaniya Thanabalasundaram
  • John D. Sinden
Part of the Neuromethods book series (NM, volume 128)


MicroRNAs (miRNAs) have been proven to regulate gene expression at post-transcriptional level and are emerging as strong mediators in neural fate determination (Ambros, Nature 431(7006):350–355, 2004). Here, we evaluated appropriate 3 three dimensional (3D) substrates to differentiate human neural stem cells (hNSCs). We identified and quantified hNSC miRNA contents by PCR array. By using computational algorithms we predicted miRNA target mRNA which correlates with hNSC differentiation and performed target validation by transfection of 3 prime untranslated regions (3′UTR) dual reporter plasmids and dual luciferase assay. Despite the inherent differences between cultures, we were able to consistently show that 3D topography promotes differentiation of hNSCs through modulation of miRNAs associated with cell proliferation and maintenance of stemness.


Clinical grade neural stem cells In vitro differentiation miRNA profiling and effects miRNA target validation Three dimensional culture 


1 W

1 Week


Two dimensional

3 W

3 Weeks


3 Prime untranslated region


Three dimensional




Basic fibroblast growth factor




Epidermal growth factor receptor




Glial fibrillary acidic protein


Human neural stem cell


Human miRNA




Microtubule-associated protein 2




Messenger RNA


Real-time reverse transcription PCR


S100 calcium binding protein B


Tubulin, beta 3 class III



This study was supported by ReNeuron (RENE.L). We acknowledge Julie Heward for helping in the preparation of hNSCs.


  1. 1.
    Ambros V (2004) The functions of animal microRNAs. Nature 431(7006):350–355. doi: 10.1038/nature02871 CrossRefPubMedGoogle Scholar
  2. 2.
    Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391(6669):806–811. doi: 10.1038/35888 CrossRefPubMedGoogle Scholar
  3. 3.
    Spivakov M, Fisher AG (2007) Epigenetic signatures of stem-cell identity. Nat Rev Genet 8(4):263–271. doi: 10.1038/nrg2046 CrossRefPubMedGoogle Scholar
  4. 4.
    Badylak SF (2002) The extracellular matrix as a scaffold for tissue reconstruction. Semin Cell Dev Biol 13(5):377–383CrossRefPubMedGoogle Scholar
  5. 5.
    Buxboim A, Discher DE (2010) Stem cells feel the difference. Nat Methods 7(9):695–697. doi: 10.1038/nmeth0910-695 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Gelain F, Bottai D, Vescovi A, Zhang S (2006) Designer self-assembling peptide nanofiber scaffolds for adult mouse neural stem cell 3-dimensional cultures. PLoS One 1:e119. doi: 10.1371/journal.pone.0000119 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Li WJ, Tuli R, Okafor C, Derfoul A, Danielson KG, Hall DJ, Tuan RS (2005) A three-dimensional nanofibrous scaffold for cartilage tissue engineering using human mesenchymal stem cells. Biomaterials 26(6):599–609. doi: 10.1016/j.biomaterials.2004.03.005 CrossRefPubMedGoogle Scholar
  8. 8.
    Lutolf MP, Gilbert PM, Blau HM (2009) Designing materials to direct stem-cell fate. Nature 462(7272):433–441. doi: 10.1038/nature08602 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Nur EKA, Ahmed I, Kamal J, Schindler M, Meiners S (2006) Three-dimensional nanofibrillar surfaces promote self-renewal in mouse embryonic stem cells. Stem Cells 24(2):426–433. doi: 10.1634/stemcells.2005-0170 CrossRefGoogle Scholar
  10. 10.
    Ortinau S, Schmich J, Block S, Liedmann A, Jonas L, Weiss DG, Helm CA, Rolfs A, Frech MJ (2010) Effect of 3D-scaffold formation on differentiation and survival in human neural progenitor cells. Biomed Eng Online 9(1):70. doi: 10.1186/1475-925X-9-70 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Saha K, Pollock JF, Schaffer DV, Healy KE (2007) Designing synthetic materials to control stem cell phenotype. Curr Opin Chem Biol 11(4):381–387. doi: 10.1016/j.cbpa.2007.05.030 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Hesse E, Hefferan TE, Tarara JE, Haasper C, Meller R, Krettek C, Lu L, Yaszemski MJ (2010) Collagen type I hydrogel allows migration, proliferation, and osteogenic differentiation of rat bone marrow stromal cells. J Biomed Mater Res A 94(2):442–449. doi: 10.1002/jbm.a.32696 PubMedPubMedCentralGoogle Scholar
  13. 13.
    Budyanto L, Goh YQ, Ooi CP (2009) Fabrication of porous poly(L-lactide) (PLLA) scaffolds for tissue engineering using liquid-liquid phase separation and freeze extraction. J Mater Sci Mater Med 20(1):105–111. doi: 10.1007/s10856-008-3545-8 CrossRefPubMedGoogle Scholar
  14. 14.
    Knight E, Murray B, Carnachan R, Przyborski S (2011) Alvetex(R): polystyrene scaffold technology for routine three dimensional cell culture. Methods Mol Biol 695:323–340. doi: 10.1007/978-1-60761-984-0_20 CrossRefPubMedGoogle Scholar
  15. 15.
    Qutachi O, Vetsch JR, Gill D, Cox H, Scurr DJ, Hofmann S, Muller R, Quirk RA, Shakesheff KM, Rahman CV (2014) Injectable and porous PLGA microspheres that form highly porous scaffolds at body temperature. Acta Biomater 10(12):5090–5098. doi: 10.1016/j.actbio.2014.08.015 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Chan BP, Leong KW (2008) Scaffolding in tissue engineering: general approaches and tissue-specific considerations. Eur Spine J 17(Suppl 4):467–479. doi: 10.1007/s00586-008-0745-3 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Bokhari M, Carnachan RJ, Przyborski SA, Cameron NR (2007) Emulsion- templated porous polymers as scaffolds for three dimensional cell culture: effect of synthesis parameters on scaffold formation and homogeneity. J Mater Chem 17:4088–4094CrossRefGoogle Scholar
  18. 18.
    Bokhari M, Carnachan RJ, Cameron NR, Przyborski SA (2007) Novel cell culture device enabling three-dimensional cell growth and improved cell function. Biochem Biophys Res Commun 354(4):1095–1100. doi: 10.1016/j.bbrc.2007.01.105 CrossRefPubMedGoogle Scholar
  19. 19.
    Krek A, Grun D, Poy MN, Wolf R, Rosenberg L, Epstein EJ, MacMenamin P, da Piedade I, Gunsalus KC, Stoffel M, Rajewsky N (2005) Combinatorial microRNA target predictions. Nat Genet 37(5):495–500. doi: 10.1038/ng1536 CrossRefPubMedGoogle Scholar
  20. 20.
    Lewis BP, Burge CB, Bartel DP (2005) Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120(1):15–20. doi: 10.1016/j.cell.2004.12.035 CrossRefPubMedGoogle Scholar
  21. 21.
    Stevanato L, Sinden JD (2014) The effects of microRNAs on human neural stem cell differentiation in two- and three-dimensional cultures. Stem Cell Res Ther 5(2):49. doi: 10.1186/scrt437 CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Pollock K, Stroemer P, Patel S, Stevanato L, Hope A, Miljan E, Dong Z, Hodges H, Price J, Sinden JD (2006) A conditionally immortal clonal stem cell line from human cortical neuroepithelium for the treatment of ischemic stroke. Exp Neurol 199(1):143–155. doi: 10.1016/j.expneurol.2005.12.011 CrossRefPubMedGoogle Scholar
  23. 23.
  24. 24.
    Maragkakis M, Alexiou P, Papadopoulos GL, Reczko M, Dalamagas T, Giannopoulos G, Goumas G, Koukis E, Kourtis K, Simossis VA, Sethupathy P, Vergoulis T, Koziris N, Sellis T, Tsanakas P, Hatzigeorgiou AG (2009) Accurate microRNA target prediction correlates with protein repression levels. BMC Bioinformatics 10:295. doi: 10.1186/1471-2105-10-295 CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Chen K, Rajewsky N (2006) Natural selection on human microRNA binding sites inferred from SNP data. Nat Genet 38(12):1452–1456. doi: 10.1038/ng1910 CrossRefPubMedGoogle Scholar
  26. 26.
  27. 27.
  28. 28.
    Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25(2):169–193CrossRefPubMedGoogle Scholar
  29. 29.
    Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3(6):1101–1108CrossRefPubMedGoogle Scholar
  30. 30.
    Stevanato L, Hicks C, Sinden JD (2015) Differentiation of a human neural stem cell line on three dimensional cultures, analysis of MicroRNA and putative target genes. J Vis Exp 98. doi: 10.3791/52410
  31. 31.
    Bentwich I (2005) Prediction and validation of microRNAs and their targets. FEBS Lett 579(26):5904–5910. doi: 10.1016/j.febslet.2005.09.040 CrossRefPubMedGoogle Scholar
  32. 32.
    Didiano D, Hobert O (2006) Perfect seed pairing is not a generally reliable predictor for miRNA-target interactions. Nat Struct Mol Biol 13(9):849–851. doi: 10.1038/nsmb1138 CrossRefPubMedGoogle Scholar
  33. 33.
    Parsons XH, Parsons JF, Moore DA (2012) Genome-scale mapping of MicroRNA signatures in human embryonic stem cell neurogenesis. Mol Med Ther 1(2). doi:  10.4172/2324-8769.1000105
  34. 34.
    Wang Y, Keys DN, Au-Young JK, Chen C (2009) MicroRNAs in embryonic stem cells. J Cell Physiol 218(2):251–255. doi: 10.1002/jcp.21607 CrossRefPubMedGoogle Scholar
  35. 35.
    Chen H, Shalom-Feuerstein R, Riley J, Zhang SD, Tucci P, Agostini M, Aberdam D, Knight RA, Genchi G, Nicotera P, Melino G, Vasa-Nicotera M (2010) miR-7 and miR-214 are specifically expressed during neuroblastoma differentiation, cortical development and embryonic stem cells differentiation, and control neurite outgrowth in vitro. Biochem Biophys Res Commun 394(4):921–927. doi: 10.1016/j.bbrc.2010.03.076 CrossRefPubMedGoogle Scholar
  36. 36.
    Weeraratne SD, Amani V, Teider N, Pierre-Francois J, Winter D, Kye MJ, Sengupta S, Archer T, Remke M, Bai AH, Warren P, Pfister SM, Steen JA, Pomeroy SL, Cho YJ (2012) Pleiotropic effects of miR-183~96~182 converge to regulate cell survival, proliferation and migration in medulloblastoma. Acta Neuropathol 123(4):539–552. doi: 10.1007/s00401-012-0969-5 CrossRefPubMedGoogle Scholar
  37. 37.
    Cioffi JA, Yue WY, Mendolia-Loffredo S, Hansen KR, Wackym PA, Hansen MR (2010) MicroRNA-21 overexpression contributes to vestibular schwannoma cell proliferation and survival. Otol Neurotol 31(9):1455–1462. doi: 10.1097/MAO.0b013e3181f20655 PubMedPubMedCentralGoogle Scholar
  38. 38.
    Cirera-Salinas D, Pauta M, Allen RM, Salerno AG, Ramirez CM, Chamorro-Jorganes A, Wanschel AC, Lasuncion MA, Morales-Ruiz M, Suarez Y, Baldan A, Esplugues E, Fernandez-Hernando C (2012) Mir-33 regulates cell proliferation and cell cycle progression. Cell Cycle 11(5):922–933. doi: 10.4161/cc.11.5.19421 CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Liu XS, Chopp M, Wang XL, Zhang L, Hozeska-Solgot A, Tang T, Kassis H, Zhang RL, Chen C, Xu J, Zhang ZG (2013) MicroRNA-17-92 cluster mediates the proliferation and survival of neural progenitor cells after stroke. J Biol Chem 288(18):12478–12488. doi: 10.1074/jbc.M112.449025 CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Porrello ER, Johnson BA, Aurora AB, Simpson E, Nam YJ, Matkovich SJ, Dorn GW 2nd, van Rooij E, Olson EN (2011) MiR-15 family regulates postnatal mitotic arrest of cardiomyocytes. Circ Res 109(6):670–679. doi: 10.1161/CIRCRESAHA.111.248880 CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Trompeter HI, Abbad H, Iwaniuk KM, Hafner M, Renwick N, Tuschl T, Schira J, Muller HW, Wernet P (2011) MicroRNAs MiR-17, MiR-20a, and MiR-106b act in concert to modulate E2F activity on cell cycle arrest during neuronal lineage differentiation of USSC. PLoS One 6(1):e16138. doi: 10.1371/journal.pone.0016138 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Lara Stevanato
    • 1
    Email author
  • Caroline Hicks
    • 1
  • Lavaniya Thanabalasundaram
    • 1
  • John D. Sinden
    • 1
  1. 1.ReNeuronPencoed Business ParkBridgendUK

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