Induced Pluripotent Stem Cells (iPSCs): An Emerging Model System for the Study of Human Neurotoxicology

  • M. Diana Neely
  • Andrew M. Tidball
  • Asad A. Aboud
  • Kevin C. Ess
  • Aaron B. Bowman
Part of the Neuromethods book series (NM, volume 56)


This chapter describes the materials and methods necessary to generate human induced pluripotent stem cells (iPSCs) from primary human fibroblasts and direct their differentiation into neural progenitor cells. Application of such methods is an emerging model for the study of neurotoxicity focused on human neurons and glia derived from specific patients. The techniques described here include primary human fibroblast culture, lentiviral/retroviral-mediated iPSC inductions, iPSC clonal expansion and maintenance, validation of pluripotency markers, and neuronal differentiation of iPSCs. Methods and applications using iPSCs are rapidly changing: here we describe the current methods used in our laboratories. The iPSC induction method featured in this chapter is based on a two-step viral transduction approach described by Dr. Shinya Yamanaka and colleagues (Cell 131:861–872, 2007) modified following the protocol of Dr. Sheng Ding and collaborators (Nat Methods 6:805–808, 2009). The neuralization method featured in this chapter is based on the method described by Lorenz Studer and colleagues (Nat Biotechnol 27:275–280, 2009). Maintenance and cryostorage methods were developed in our lab by optimizing a combination of approaches described in the literature. This chapter is not meant to be comprehensive, but instead focuses on the core competencies needed to begin working with human iPSCs and neuralization of these cells for toxicological studies.

Key words

Induced pluripotent stem cells Human models of neurotoxicity Neuronal differentiation Patient-derived fibroblasts 



We would like to thank Angela Ellen Tidball for the artwork and illustration of Fig. 1 of this chapter. Also, we are grateful to Dr. Lorenz Studer and Stuart Chambers for personal communications on implementing their published neuralization protocols. AMT was supported by the Vanderbilt Brain Institute. KCE was supported by a Doris Duke Clinical Scientist Development Award, NIH/NINDS K08NS050484, and the Tuberous Sclerosis Alliance. This work was further supported by a Hobbs Discovery Award from the Vanderbilt Kennedy Center (KCE, ABB), an equipment grant from the Vanderbilt Institute for Clinical and Translational Research 1UL 1RR024975 NCRR/NIH (KCE, ABB), core support from NIH NICHD grant P30HD15052 (ABB), and research support from the NIH/NIEHS grant RO1ES016931 (ABB).


  1. 1.
    Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861–872PubMedCrossRefGoogle Scholar
  2. 2.
    Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science 318:1917–1920PubMedCrossRefGoogle Scholar
  3. 3.
    Park IH, Zhao R, West JA, Yabuuchi A, Huo H, Ince TA, Lerou PH, Lensch MW, Daley GQ (2008) Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451:141–146PubMedCrossRefGoogle Scholar
  4. 4.
    Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, Ichisaka T, Aoi T, Okita K, Mochiduki Y, Takizawa N, Yamanaka S (2008) Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol 26:101–106PubMedCrossRefGoogle Scholar
  5. 5.
    Chambers SM, Fasano CA, Papapetrou EP, Tomishima M, Sadelain M, Studer L (2009) Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol 27:275–280PubMedCrossRefGoogle Scholar
  6. 6.
    Aasen T, Raya A, Barrero MJ, Garreta E, Consiglio A, Gonzalez F, Vassena R, Bilic J, Pekarik V, Tiscornia G, Edel M, Boue S, Belmonte JCI (2008) Efficient and rapid gene­ration of induced pluripotent stem cells from human keratinocytes. Nat Biotechnol 26:1276–1284PubMedCrossRefGoogle Scholar
  7. 7.
    Takahashi K, Okita K, Nakagawa M, Yamanaka S (2007) Induction of pluripotent stem cells from fibroblast cultures. Nat Protoc 2:3081–3089PubMedCrossRefGoogle Scholar
  8. 8.
    Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663–676PubMedCrossRefGoogle Scholar
  9. 9.
    Lin T, Ambasudhan R, Yuan X, Li W, Hilcove S, Abujarour R, Lin X, Hahm HS, Hao E, Hayek A, Ding S (2009) A chemical platform for improved induction of human iPSCs. Nat Methods 6:805–808PubMedCrossRefGoogle Scholar
  10. 10.
    Hockemeyer D, Soldner F, Cook EG, Gao Q, Mitalipova M, Jaenisch R (2008) A drug-inducible system for direct reprogramming of human somatic cells to pluripotency. Cell Stem Cell 3:346–353PubMedCrossRefGoogle Scholar
  11. 11.
    Hong H, Takahashi K, Ichisaka T, Aoi T, Kanagawa O, Nakagawa M, Okita K, Yamanaka S (2009) Suppression of induced pluripotent stem cell generation by the p53-p21 pathway. Nature 460:1132–1135PubMedCrossRefGoogle Scholar
  12. 12.
    Kaji K, Norrby K, Paca A, Mileikovsky M, Mohseni P, Woltjen K (2009) Virus-free induc­tion of pluripotency and subsequent excision of reprogramming factors. Nature 458:771–775PubMedCrossRefGoogle Scholar
  13. 13.
    Okita K, Nakagawa M, Hyenjong H, Ichisaka T, Yamanaka S (2008) Generation of mouse induced pluripotent stem cells without viral vectors. Science 322:949–953PubMedCrossRefGoogle Scholar
  14. 14.
    Woltjen K, Michael IP, Mohseni P, Desai R, Mileikovsky M, Hämäläinen R, Cowling R, Wang W, Liu P, Gertsenstein M, Kaji K, Sung H-K, Nagy A (2009) piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature 458:766–770PubMedCrossRefGoogle Scholar
  15. 15.
    Yoshida Y, Takahashi K, Okita K, Ichisaka T, Yamanaka S (2009) Hypoxia enhances the generation of induced pluripotent stem cells. Cell Stem Cell 5:237–241PubMedCrossRefGoogle Scholar
  16. 16.
    Ohnuki M, Takahashi K, Yamanaka S (2009) Generation and characterization of human induced pluripotent stem cells. Curr Protoc Stem Cell Biol Chapter 4:Unit 4A.2Google Scholar
  17. 17.
    Chan EM, Ratanasirintrawoot S, Park IH, Manos PD, Loh YH, Huo H, Miller JD, Hartung O, Rho J, Ince TA, Daley GQ, Schlaeger TM (2009) Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells. Nat Biotechnol 27:1033–1037PubMedCrossRefGoogle Scholar
  18. 18.
    Hanna J, Saha K, Pando B, van Zon J, Lengner CJ, Creyghton MP, van Oudenaarden A, Jaenisch R (2009) Direct cell reprogramming is a stochastic process amenable to acceleration. Nature 462:595–601PubMedCrossRefGoogle Scholar
  19. 19.
    Friedrich G, Soriano P (1993) Insertional mutagenesis by retroviruses and promoter traps in embryonic stem cells. Methods Enzymol 225:681–701PubMedCrossRefGoogle Scholar
  20. 20.
    Sjögren-Jansson E, Zetterström M, Moya K, Lindqvist J, Strehl R, Eriksson PS (2005) Large-scale propagation of four undifferentiated human embryonic stem cell lines in a feeder-free culture system. Dev Dyn 233:1304–1314PubMedCrossRefGoogle Scholar
  21. 21.
    Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD, Carpenter MK (2001) Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 19:971–974PubMedCrossRefGoogle Scholar
  22. 22.
    Amit M, Carpenter MK, Inokuma MS, Chiu CP, Harris CP, Waknitz MA, Itskovitz-Eldor J, Thomson JA (2000) Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev Biol 227:271–278PubMedCrossRefGoogle Scholar
  23. 23.
    Mollamohammadi S, Taei A, Pakzad M, Totonchi M, Seifinejad A, Masoudi N, Baharvand H (2009) A simple and efficient cryopreservation method for feeder-free dissociated human induced pluripotent stem cells and human embryonic stem cells. Hum Reprod 24:2468–2476PubMedCrossRefGoogle Scholar
  24. 24.
    Kleinman HK, McGarvey ML, Liotta LA, Robey PG, Tryggvason K, Martin GR (1982) Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma. Biochemistry 21:6188–6193PubMedCrossRefGoogle Scholar
  25. 25.
    Bissell DM, Arenson DM, Maher JJ, Roll FJ (1987) Support of cultured hepatocytes by a laminin-rich gel. Evidence for a functionally significant subendothelial matrix in normal rat liver. J Clin Invest 79:801–812PubMedCrossRefGoogle Scholar
  26. 26.
    Watanabe K, Ueno M, Kamiya D, Nishiyama A, Matsumura M, Wataya T, Takahashi JB, Nishikawa S, Nishikawa S-i, Muguruma K, Sasai Y (2007) A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotechnol 25:681–686PubMedCrossRefGoogle Scholar
  27. 27.
    O’Connor MD, Kardel MD, Iosfina I, Youssef D, Lu M, Li MM, Vercauteren S, Nagy A, Eaves CJ (2008) Alkaline phosphatase-positive colony formation is a sensitive specific, and quantitative indicator of undifferentiated human embryonic stem cells. Stem Cells 26:1109–1116PubMedCrossRefGoogle Scholar
  28. 28.
    Silva J, Nichols J, Theunissen TW, Guo G, van Oosten AL, Barrandon O, Wray J, Yamanaka S, Chambers I, Smith A (2009) Nanog is the gateway to the pluripotent ground state. Cell 138:722–737PubMedCrossRefGoogle Scholar
  29. 29.
    Lutfalla G, Uze G (2006) Performing quantitative reverse-transcribed polymerase chain reaction experiments. Methods Enzymol 410:386–400PubMedCrossRefGoogle Scholar
  30. 30.
    Wong ML, Medrano JF (2005) Real-time PCR for mRNA quantitation. Biotechniques 39:75–85PubMedCrossRefGoogle Scholar
  31. 31.
    Ungrin M, O’Connor M, Eaves C, Zandstra PW (2007) Phenotypic analysis of human embryonic stem cells. Curr Protoc Stem Cell Biol Chapter 1:Unit 1B.3Google Scholar
  32. 32.
    Totonchi M, Taei A, Seifinejad A, Tabebordbar M, Rassouli H, Farrokhi A, Gourabi H, Aghdami N, Hosseini-Salekdeh G, Baharvand H (2010) Feeder- and serum-free establishment and expansion of human induced pluripotent stem cells. Int J Dev Biol 54(5):877–886.PubMedCrossRefGoogle Scholar
  33. 33.
    Elkabetz Y, Panagiotakos G, Al Shamy G, Socci ND, Tabar V, Studer L (2008) Human ES cell-derived neural rosettes reveal a functionally distinct early neural stem cell stage. Genes Dev 22:152–165PubMedCrossRefGoogle Scholar
  34. 34.
    Lee H, Shamy GA, Elkabetz Y, Schofield CM, Harrsion NL, Panagiotakos G, Socci ND, Tabar V, Studer L (2007) Directed differentiation and transplantation of human embryonic stem cell-derived motoneurons. Stem Cells 25:1931–1939PubMedCrossRefGoogle Scholar
  35. 35.
    Smith JR, Vallier L, Lupo G, Alexander M, Harris WA, Pedersen RA (2008) Inhibition of Activin/Nodal signaling promotes specification of human embryonic stem cells into neuroectoderm. Dev Biol 313:107–117PubMedCrossRefGoogle Scholar
  36. 36.
    Valenzuela DM, Economides AN, Rojas E, Lamb TM, Nuñez L, Jones P, Lp NY, Espinosa R, Brannan CI, Gilbert DJ (1995) Identification of mammalian noggin and its expression in the adult nervous system. J Neurosci 15:6077–6084PubMedGoogle Scholar
  37. 37.
    Li X-J, Du Z-W, Zarnowska ED, Pankratz M, Hansen LO, Pearce RA, Zhang S-C (2005) Specification of motoneurons from human embryonic stem cells. Nat Biotechnol 23:215–221PubMedCrossRefGoogle Scholar
  38. 38.
    Perrier AL, Tabar V, Barberi T, Rubio ME, Bruses J, Topf N, Harrison NL, Studer L (2004) Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc Natl Acad Sci U S A 101:12543–12548PubMedCrossRefGoogle Scholar
  39. 39.
    Hu B-Y, Zhang S-C (2009) Differentiation of spinal motor neurons from pluripotent human stem cells. Nat Protoc 4:1295–1304PubMedCrossRefGoogle Scholar
  40. 40.
    Aubry L, Bugi A, Lefort N, Rousseau F, Peschanski M, Perrier AL (2008) Striatal progenitors derived from human ES cells mature into DARPP32 neurons in vitro and in quinolinic acid-lesioned rats. Proc Natl Acad Sci U S A 105:16707–16712PubMedCrossRefGoogle Scholar
  41. 41.
    Zhang X-Q, Zhang S-C (2010) Differentiation of neural precursors and dopaminergic ­neurons from human embryonic stem cells. Methods Mol Biol 584:355–366PubMedCrossRefGoogle Scholar
  42. 42.
    Carpenter M, Inokuma M, Denham J, Mujtaba T, Chiu C, Rao M (2001) Enrichment of neurons and neural precursors from human embryonic stem cells. Exp Neurol 172:383–397PubMedCrossRefGoogle Scholar
  43. 43.
    Gaspard N, Bouschet T, Hourez R, Dimidschstein J, Naeije G, Van Den Ameele J, Espuny-Camacho I, Herpoel A, Passante L, Schiffmann SN, Gaillard A, Vanderhaeghen P (2008) An intrinsic mechanism of corticogenesis from embryonic stem cells. Nature 455:351–357PubMedCrossRefGoogle Scholar
  44. 44.
    Goldstein RS, Pomp O, Brokhman I, Ziegler L (2010) Generation of neural crest cells and peri­pheral sensory neurons from human embryonic stem cells. Methods Mol Biol 584:283–300PubMedCrossRefGoogle Scholar
  45. 45.
    Ozolek JA, Jane EP, Esplen JE, Petrosko P, Wehn AK, Erb TM, Mucko SE, Cote LC, Sammak PJ (2010) In vitro neural differentiation of human embryonic stem cells using a low-density mouse embryonic fibroblast feeder protocol. Methods Mol Biol 584:71–95PubMedCrossRefGoogle Scholar
  46. 46.
    Fasano CA, Chambers SM, Lee G, Tomishima MJ, Studer L (2010) Efficient derivation of functional floor plate tissue from human embryonic stem cells. Cell Stem Cell 6:336–347PubMedCrossRefGoogle Scholar
  47. 47.
    Cooper O, Hargus G, Deleidi M, Blak A, Osborn T, Marlow E, Lee K, Levy A, Perez-Torres E, Yow A, Isacson O (2010) Differen­tiation of human ES and Parkinson’s disease iPS cells into ventral midbrain dopaminergic neurons requires a high activity form of SHH, FGF8a and specific regionalization by retinoic acid. Mol Cell Neurosci 45:258–266PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • M. Diana Neely
  • Andrew M. Tidball
  • Asad A. Aboud
  • Kevin C. Ess
  • Aaron B. Bowman
    • 1
  1. 1.Department of Neurology, Vanderbilt Kennedy Center for Research on Human DevelopmentVanderbilt University Medical CenterNashvilleUSA

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