Development and characterization of a human embryonic stem cell-derived 3D neural tissue model for neurotoxicity testing

Toxicology in Vitro

Volumn 38, Issue , 2017, Pages 124-135

  Sandstrom J ^a   Eggermann, E ^b   Charvet, I ^b   Roux, A ^b   Toni, N ^a   Greggio, C ^a   Broyer, A ^a   Monnet Tschudi, F ^a   Stoppini, L ^b  

^a UNIVERSITY OF LAUSANNE   (Switzerland)

^b Swiss Centre for Applied Human Toxicology   (Switzerland)

Author keywords

3D cultures; Astrocytes; Human embryonic stem cells; Neurons; Neurotoxicity testing; Oligodendrocytes; Spontaneous electrical activity

Indexed keywords

4 AMINOBUTYRIC ACID B RECEPTOR; ACETYLCHOLINESTERASE; BETA TUBULIN; DOUBLECORTIN; GLIAL FIBRILLARY ACIDIC PROTEIN; MESSENGER RNA; METHYLMERCURY; MICROTUBULE ASSOCIATED PROTEIN 2; MYELIN; MYELIN BASIC PROTEIN; N METHYL DEXTRO ASPARTIC ACID RECEPTOR; NEUROFILAMENT PROTEIN; NOTCH1 RECEPTOR; OLIGODENDROCYTE TRANSCRIPTION FACTOR 2; PARAQUAT; TRANSCRIPTION FACTOR SOX10; TRIMETHYLTIN; TYROSINE 3 MONOOXYGENASE; VESICULAR INHIBITORY AMINO ACID TRANSPORTER; VIMENTIN; IBUPROFEN; METHYLMERCURIC CHLORIDE; METHYLMERCURY DERIVATIVE; TRIMETHYLTIN CHLORIDE;

ARTICLE; ASTROCYTE; AXON; CELL MATURATION; CONTROLLED STUDY; EMBRYONIC STEM CELL; HUMAN; HUMAN CELL; HUMAN TISSUE; IN VITRO STUDY; MACROGLIA; MYELIN SHEATH; NERVE CELL CULTURE; NERVE CELL DIFFERENTIATION; NERVOUS SYSTEM ELECTROPHYSIOLOGY; NERVOUS TISSUE; NEURAL STEM CELL; NEUROTOXICITY; OLIGODENDROGLIA; PROTEIN EXPRESSION; SYNAPSE; TOXICITY TESTING; CELL CULTURE TECHNIQUE; CELL DIFFERENTIATION; CELL LINE; CYTOLOGY; DRUG EFFECTS; GENE EXPRESSION; HUMAN EMBRYONIC STEM CELL; METABOLISM; MULTICELLULAR SPHEROID; TISSUE CULTURE TECHNIQUE; ULTRASTRUCTURE;

ASTROCYTES; CELL CULTURE TECHNIQUES; CELL DIFFERENTIATION; CELL LINE; GENE EXPRESSION; HUMAN EMBRYONIC STEM CELLS; HUMANS; IBUPROFEN; METHYLMERCURY COMPOUNDS; NEURAL STEM CELLS; OLIGODENDROGLIA; PARAQUAT; RNA, MESSENGER; SPHEROIDS, CELLULAR; TISSUE CULTURE TECHNIQUES; TRIMETHYLTIN COMPOUNDS;

EID: 85006972234     PISSN: 08872333     EISSN: 18793177     Source Type: Journal    
DOI: 10.1016/j.tiv.2016.10.001     Document Type: Article

References (74)

1. Ajami, B., Bennett, J.L., Krieger, C., McNagny, K.M., Rossi, F.M., Infiltrating monocytes trigger EAE progression, but do not contribute to the resident microglia pool. Nat. Neurosci. 14 (2011), 1142–1149.
2. Alepee, N., Bahinski, A., Daneshian, M., De Wever, B., Fritsche, E., Goldberg, A., Hansmann, J., Hartung, T., Haycock, J., Hogberg, H., Hoelting, L., Kelm, J.M., Kadereit, S., McVey, E., Landsiedel, R., Leist, M., Lubberstedt, M., Noor, F., Pellevoisin, C., Petersohn, D., Pfannenbecker, U., Reisinger, K., Ramirez, T., Rothen-Rutishauser, B., Schafer-Korting, M., Zeilinger, K., Zurich, M.G., State-of-the-art of 3D cultures (organs-on-a-chip) in safety testing and pathophysiology. ALTEX 31 (2014), 441–477.
3. Bal-Price, A., Crofton, K.M., Leist, M., Allen, S., Arand, M., Buetler, T., Delrue, N., FitzGerald, R.E., Hartung, T., Heinonen, T., Hogberg, H., Bennekou, S.H., Lichtensteiger, W., Oggier, D., Paparella, M., Axelstad, M., Piersma, A., Rached, E., Schilter, B., Schmuck, G., Stoppini, L., Tongiorgi, E., Tiramani, M., Monnet-Tschudi, F., Wilks, M.F., Ylikomi, T., Fritsche, E., International STakeholder NETwork (ISTNET): creating a developmental neurotoxicity (DNT) testing road map for regulatory purposes. Arch. Toxicol. 89 (2015), 269–287.
4. Baumann, J., Barenys, M., Gassmann, K., Fritsche, E., Comparative human and rat “neurosphere assay” for developmental neurotoxicity testing. Curr. Protoc. Toxicol. 59 (2014), 12 21 11–12 21 24.
5. Bramanti, V., Tomassoni, D., Avitabile, M., Amenta, F., Avola, R., Biomarkers of glial cell proliferation and differentiation in culture. Front. Biosci. (Schol. Ed.) 2 (2010), 558–570.
6. Castello, P.R., Drechsel, D.A., Patel, M., Mitochondria are a major source of paraquat-induced reactive oxygen species production in the brain. J. Biol. Chem. 282 (2007), 14186–14193.
7. Chen, K.G., Mallon, B.S., Hamilton, R.S., Kozhich, O.A., Park, K., Hoeppner, D.J., Robey, P.G., McKay, R.D., Non-colony type monolayer culture of human embryonic stem cells. Stem Cell Res. 9 (2012), 237–248.
8. Choi, S.H., Kim, Y.H., Hebisch, M., Sliwinski, C., Lee, S., D'Avanzo, C., Chen, H., Hooli, B., Asselin, C., Muffat, J., Klee, J.B., Zhang, C., Wainger, B.J., Peitz, M., Kovacs, D.M., Woolf, C.J., Wagner, S.L., Tanzi, R.E., Kim, D.Y., A three-dimensional human neural cell culture model of Alzheimer's disease. Nature 515 (2014), 274–278.
9. Connors, B.W., Gutnick, M.J., Intrinsic firing patterns of diverse neocortical neurons. Trends Neurosci. 13 (1990), 99–104.
10. Drechsel, D.A., Patel, M., Role of reactive oxygen species in the neurotoxicity of environmental agents implicated in Parkinson's disease. Free Radic. Biol. Med. 44 (2008), 1873–1886.
11. Dubois-Dauphin, M.L., Toni, N., Julien, S.D., Charvet, I., Sundstrom, L.E., Stoppini, L., The long-term survival of in vitro engineered nervous tissue derived from the specific neural differentiation of mouse embryonic stem cells. Biomaterials 31 (2010), 7032–7042.
12. Eng, L.F., Ghirnikar, R.S., Lee, Y.L., Glial fibrillary acidic protein: GFAP-thirty-one years (1969–2000). NeurochemRes 25 (2000), 1439–1451.
13. Fancy, S.P., Chan, J.R., Baranzini, S.E., Franklin, R.J., Rowitch, D.H., Myelin regeneration: a recapitulation of development?. Annu. Rev. Neurosci. 34 (2011), 21–43.
14. Gassmann, K., Abel, J., Bothe, H., Haarmann-Stemmann, T., Merk, H.F., Quasthoff, K.N., Rockel, T.D., Schreiber, T., Fritsche, E., Species-specific differential AhR expression protects human neural progenitor cells against developmental neurotoxicity of PAHs. Environ. Health Perspect. 118 (2010), 1571–1577.
15. Gomez Perdiguero, E., Schulz, C., Geissmann, F., Development and homeostasis of “resident” myeloid cells: the case of the microglia. Glia 61 (2013), 112–120.
16. Gritti, A., Bonfanti, L., Neuronal-glial interactions in central nervous system neurogenesis: the neural stem cell perspective. Neuron Glia Biol. 3 (2007), 309–323.
17. Hoelting, L., Scheinhardt, B., Bondarenko, O., Schildknecht, S., Kapitza, M., Tanavde, V., Tan, B., Lee, Q.Y., Mecking, S., Leist, M., Kadereit, S., A 3-dimensional human embryonic stem cell (hESC)-derived model to detect developmental neurotoxicity of nanoparticles. Arch. Toxicol. 87 (2013), 721–733.
18. Hogberg, H.T., Bressler, J., Christian, K.M., Harris, G., Makri, G., O'Driscoll, C., Pamies, D., Smirnova, L., Wen, Z., Hartung, T., Toward a 3D model of human brain development for studying gene/environment interactions. Stem Cell Res. Ther., 4(Suppl 1), 2013, S4.
19. Honegger, P., Monnet-Tschudi, F., Aggregating neural cell cultures. Fedoroff, S., Richardson, A., (eds.) Protocols for Neural Cell Cultures, 3 edn, 2001, Humana Press, Inc., Totowa, 199–218.
20. Honegger, P., Zurich, M., Preparation and use of serum-free aggregating brain cell cultures for routine neurotoxicity screening. Aschner, M., Sunol, C., Bal-Price, A., (eds.) Cell Culture Techniques, 2011, Humana Press, New York, 105–128.
21. Honegger, P., Lenoir, D., Favrod, P., Growth and differentiation of aggregating fetal brain cells in a serum-free defined medium. Nature 282 (1979), 305–308.
22. Honegger, P., Defaux, A., Monnet-Tschudi, F., Zurich, M.G., Preparation, maintenance, and use of serum-free aggregating brain cell cultures. Methods Mol. Biol. 758 (2011), 81–97.
23. Jiang, X., Nardelli, J., Cellular and molecular introduction to brain development. Neurobiol. Dis. 92 (2016), 3–17.
24. Kim, Y.H., Choi, S.H., D'Avanzo, C., Hebisch, M., Sliwinski, C., Bylykbashi, E., Washicosky, K.J., Klee, J.B., Brustle, O., Tanzi, R.E., Kim, D.Y., A 3D human neural cell culture system for modeling Alzheimer's disease. Nat. Protoc. 10 (2015), 985–1006.
25. Knight, E., Przyborski, S., Advances in 3D cell culture technologies enabling tissue-like structures to be created in vitro. J. Anat. 227 (2015), 746–756.
26. Koh, J.Y., Choi, D.W., Quantitative determination of glutamate mediated cortical neuronal injury in cell culture by lactate dehydrogenase efflux assay. J. Neurosci. Methods 20 (1987), 83–90.
27. Krug, A.K., Kolde, R., Gaspar, J.A., Rempel, E., Balmer, N.V., Meganathan, K., Vojnits, K., Baquie, M., Waldmann, T., Ensenat-Waser, R., Jagtap, S., Evans, R.M., Julien, S., Peterson, H., Zagoura, D., Kadereit, S., Gerhard, D., Sotiriadou, I., Heke, M., Natarajan, K., Henry, M., Winkler, J., Marchan, R., Stoppini, L., Bosgra, S., Westerhout, J., Verwei, M., Vilo, J., Kortenkamp, A., Hescheler, J., Hothorn, L., Bremer, S., van Thriel, C., Krause, K.H., Hengstler, J.G., Rahnenfuhrer, J., Leist, M., Sachinidis, A., Human embryonic stem cell-derived test systems for developmental neurotoxicity: a transcriptomics approach. Arch. Toxicol. 87 (2013), 123–143.
28. Lancaster, M.A., Knoblich, J.A., Generation of cerebral organoids from human pluripotent stem cells. Nat. Protoc. 9 (2014), 2329–2340.
29. Lancaster, M.A., Renner, M., Martin, C.A., Wenzel, D., Bicknell, L.S., Hurles, M.E., Homfray, T., Penninger, J.M., Jackson, A.P., Knoblich, J.A., Cerebral organoids model human brain development and microcephaly. Nature 501 (2013), 373–379.
30. Larionov, A., Krause, A., Miller, W., A standard curve based method for relative real time PCR data processing. BMC Bioinf., 6, 2005, 62.
31. Lee, T.C., Lee, T.H., Huang, Y.H., Chang, N.K., Lin, Y.J., Chien, P.W., Yang, W.H., Lin, M.H., Comparison of surface markers between human and rabbit mesenchymal stem cells. PLoS One, 9, 2014, e111390.
32. Leist, M., Bremer, S., Brundin, P., Hescheler, J., Kirkeby, A., Krause, K.H., Poerzgen, P., Puceat, M., Schmidt, M., Schrattenholz, A., Zak, N.B., Hentze, H., The biological and ethical basis of the use of human embryonic stem cells for in vitro test systems or cell therapy. ALTEX 25 (2008), 163–190.
33. Leist, M., Hartung, T., Nicotera, P., The dawning of a new age of toxicology. ALTEX 25 (2008), 103–114.
34. Li, H., Wijekoon, A., Leipzig, N.D., 3D differentiation of neural stem cells in macroporous photopolymerizable hydrogel scaffolds. PLoS One, 7, 2012, e48824.
35. Liu, M.C., Liu, X.Q., Wang, W., Shen, X.F., Che, H.L., Guo, Y.Y., Zhao, M.G., Chen, J.Y., Luo, W.J., Involvement of microglia activation in the lead induced long-term potentiation impairment. PLoS One, 7, 2012, e43924.
36. Liu, Y., Zeng, X., Hui, Y., Zhu, C., Wu, J., Taylor, D.H., Ji, J., Fan, W., Huang, Z., Hu, J., Activation of alpha7 nicotinic acetylcholine receptors protects astrocytes against oxidative stress-induced apoptosis: implications for Parkinson's disease. Neuropharmacology 91 (2015), 87–96.
37. Monnet-Tschudi, F., Zurich, M.G., Honegger, P., Evaluation of the toxicity of different metal compounds in the developing brain using aggregating cell cultures as a model. Toxicol. in Vitro 7 (1993), 335–339.
38. Monnet-Tschudi, F., Zurich, M.G., Pithon, E., van Melle, G., Honegger, P., Microglial responsiveness as a sensitive marker for trimethyltin (TMT) neurotoxicity. Brain Res. 690 (1995), 8–14.
39. Monnet-Tschudi, F., Zurich, M.G., Honegger, P., Comparison of the developmental effects of two mercury compounds on glial cells and neurons in aggregate cultures of rat telencephalon. Brain Res. 741 (1996), 52–59.
40. Monnet-Tschudi, F., Zurich, M.G., Honegger, P., Neurotoxicant-induced inflammatory response in three-dimensional brain cell cultures. Hum. Exp. Toxicol. 26 (2007), 339–346.
41. Monnet-Tschudi, F., Defaux, A., Zurich, M.-G., Probable involvement of heavy metal-induced neuroinflammation in neurodegeneration. Huang, S., (eds.) Heavy Metals and Neurodegeneration, 2010, Kerala, India, Research Signpost.
42. Moscona, A.A., Rotation-mediated histogenetic aggregation of dissociated cells: a quantifiable approachh to cell interactions in vitro. ExpCell. Res. 22 (1961), 455–475.
43. Pamies, D., Hartung, T., Hogberg, H.T., Biological and medical applications of a brain-on-a-chip. Exp. Biol. Med. (Maywood) 239 (2014), 1096–1107.
44. Pasca, A.M., Sloan, S.A., Clarke, L.E., Tian, Y., Makinson, C.D., Huber, N., Kim, C.H., Park, J.Y., O'Rourke, N.A., Nguyen, K.D., Smith, S.J., Huguenard, J.R., Geschwind, D.H., Barres, B.A., Pasca, S.P., Functional cortical neurons and astrocytes from human pluripotent stem cells in 3D culture. Nat. Methods 12 (2015), 671–678.
45. Philippot, G., Nyberg, F., Gordh, T., Fredriksson, A., Viberg, H., Short-term exposure and long-term consequences of neonatal exposure to Delta(9)-tetrahydrocannabinol (THC) and ibuprofen in mice. Behav. Brain Res. 307 (2016), 137–144.
46. Pinto, J.G., Jones, D.G., Murphy, K.M., Comparing development of synaptic proteins in rat visual, somatosensory, and frontal cortex. Front. Neural Circ., 7, 2013, 97.
47. Pozniak, C.D., Langseth, A.J., Dijkgraaf, G.J., Choe, Y., Werb, Z., Pleasure, S.J., Sox10 directs neural stem cells toward the oligodendrocyte lineage by decreasing suppressor of fused expression. Proc. Natl. Acad. Sci. U. S. A. 107 (2010), 21795–21800.
48. Prieto, P., Kinsner-Ovaskainen, A., Stanzel, S., Albella, B., Artursson, P., Campillo, N., Cecchelli, R., Cerrato, L., Diaz, L., Di Consiglio, E., Guerra, A., Gombau, L., Herrera, G., Honegger, P., Landry, C., O'Connor, J.E., Paez, J.A., Quintas, G., Svensson, R., Turco, L., Zurich, M.G., Zurbano, M.J., Kopp-Schneider, A., The value of selected in vitro and in silico methods to predict acute oral toxicity in a regulatory context: results from the European project ACuteTox. Toxicol. in Vitro 27 (2013), 1357–1376.
49. Purisai, M.G., McCormack, A.L., Cumine, S., Li, J., Isla, M.Z., Di Monte, D.A., Microglial activation as a priming event leading to paraquat-induced dopaminergic cell degeneration. Neurobiol. Dis. 25 (2007), 392–400.
50. Qian, X., Nguyen, H.N., Song, M.M., Hadiono, C., Ogden, S.C., Hammack, C., Yao, B., Hamersky, G.R., Jacob, F., Zhong, C., Yoon, K.J., Jeang, W., Lin, L., Li, Y., Thakor, J., Berg, D.A., Zhang, C., Kang, E., Chickering, M., Nauen, D., Ho, C.Y., Wen, Z., Christian, K.M., Shi, P.Y., Maher, B.J., Wu, H., Jin, P., Tang, H., Song, H., Ming, G.L., Brain-region-specific organoids using mini-bioreactors for modeling ZIKV exposure. Cell 165 (2016), 1238–1254.
51. Ramakers, C., Ruijter, J.M., Deprez, R.H., Moorman, A.F., Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci. Lett. 339 (2003), 62–66.
52. Rey, H.G., Pedreira, C., Quian Quiroga, R., Past, present and future of spike sorting techniques. Brain Res. Bull. 119 (2015), 106–117.
53. Rovida, C., Vivier, M., Garthoff, B., Hescheler, J., ESNATS conference - the use of human embryonic stem cells for novel toxicity testing approaches. Altern. Lab. Anim: ATLA 42 (2014), 97–113.
54. Schwartz, M.P., Hou, Z., Propson, N.E., Zhang, J., Engstrom, C.J., Santos Costa, V., Jiang, P., Nguyen, B.K., Bolin, J.M., Daly, W., Wang, Y., Stewart, R., Page, C.D., Murphy, W.L., Thomson, J.A., Human pluripotent stem cell-derived neural constructs for predicting neural toxicity. Proc. Natl. Acad. Sci. U. S. A. 112 (2015), 12516–12521.
55. Shea, T.B., Beermann, M.L., Respective roles of neurofilaments, microtubules, MAP1B, and tau in neurite outgrowth and stabilization. Mol. Biol. Cell 5 (1994), 863–875.
56. Shinde, V., Klima, S., Sureshkumar, P.S., Meganathan, K., Jagtap, S., Rempel, E., Rahnenfuhrer, J., Hengstler, J.G., Waldmann, T., Hescheler, J., Leist, M., Sachinidis, A., Human pluripotent stem cell based developmental toxicity assays for chemical safety screening and systems biology data generation. J. Vis. Exp.: JoVE, 2015, e52333.
57. Singh, R.K., Mallela, R.K., Cornuet, P.K., Reifler, A.N., Chervenak, A.P., West, M.D., Wong, K.Y., Nasonkin, I.O., Characterization of three-dimensional retinal tissue derived from human embryonic stem cells in adherent monolayer cultures. Stem Cells Dev. 24 (2015), 2778–2795.
58. Stolt, C.C., Rehberg, S., Ader, M., Lommes, P., Riethmacher, D., Schachner, M., Bartsch, U., Wegner, M., Terminal differentiation of myelin-forming oligodendrocytes depends on the transcription factor Sox10. Genes Dev. 16 (2002), 165–170.
59. Stoppini, L., von Tobel J, S., Charvet, I., M-G, Z., Sundstom, L., Monnet-Tschudi, F., 3D in vitro models for developmental neurotoxicity (DNT). Slikker, W., Chang, W., (eds.) Neural Cell Biology, 8, 2016.
60. Tieng, V., Stoppini, L., Villy, S., Fathi, M., Dubois-Dauphin, M., Krause, K.H., Engineering of midbrain organoids containing long-lived dopaminergic neurons. Stem Cells Dev. 23 (2014), 1535–1547.
61. Uenishi, G., Theisen, D., Lee, J.H., Kumar, A., Raymond, M., Vodyanik, M., Swanson, S., Stewart, R., Thomson, J., Slukvin, I., Tenascin C promotes hematoendothelial development and T lymphoid commitment from human pluripotent stem cells in chemically defined conditions. Stem Cell Rep. 3 (2014), 1073–1084.
62. Verney, C., Phenotypic expression of monoamines and GABA in the early development of human telencephalon, transient or not transient. J. Chem. Neuroanat. 26 (2003), 283–292.
63. Wilson, P.G., Stice, S.S., Development and differentiation of neural rosettes derived from human embryonic stem cells. Stem Cell Rev. 2 (2006), 67–77.
64. Yang, S.T., Zhang, X., Wen, Y., Microbioreactors for high-throughput cytotoxicity assays. Curr. Opin. Drug Discov. Devel. 11 (2008), 111–127.
65. Yi, Y., Park, J., Lim, J., Lee, C.J., Lee, S.H., Central nervous system and its disease models on a Chip. Trends Biotechnol. 33 (2015), 762–776.
66. Zhang, Y., He, K., Wang, F., Li, X., Liu, D., Notch-1 signaling regulates astrocytic proliferation and activation after hypoxia exposure. Neurosci. Lett. 603 (2015), 12–18.
67. Zhang, Z.N., Freitas, B.C., Qian, H., Lux, J., Acab, A., Trujillo, C.A., Herai, R.H., Nguyen Huu, V.A., Wen, J.H., Joshi-Barr, S., Karpiak, J.V., Engler, A.J., Fu, X.D., Muotri, A.R., Almutairi, A., Layered hydrogels accelerate iPSC-derived neuronal maturation and reveal migration defects caused by MeCP2 dysfunction. Proc. Natl. Acad. Sci. U. S. A. 113 (2016), 3185–3190.
68. Zhou, F., Wu, J.Y., Sun, X.L., Yao, H.H., Ding, J.H., Hu, G., Iptakalim alleviates rotenone-induced degeneration of dopaminergic neurons through inhibiting microglia-mediated neuroinflammation. Neuropsychopharmacology 32 (2007), 2570–2580.
69. Zhou, S., Szczesna, K., Ochalek, A., Kobolak, J., Varga, E., Nemes, C., Chandrasekaran, A., Rasmussen, M., Cirera, S., Hyttel, P., Dinnyes, A., Freude, K.K., Avci, H.X., Neurosphere based differentiation of human iPSC improves astrocyte differentiation. Stem Cells Int., 2016, 2016, 4937689.
70. Zurich, M.-G., Eskes, C., Honegger, P., Bérode, M., Monnet-Tschudi, F., Maturation-dependent neurotoxicity of lead aceate in vitro: implication of glial reactions. J. Neurosci. Res. 70 (2002), 108–116.
71. Zurich, M.-G., Monnet-Tschudi, F., Costa, L.C., Schilter, B., Honegger, P., Aggregating brain cell cultures for neurotoxicological studies. Tiffany-Castiglioni, E., (eds.) In vitro Neurotoxicology: Principles and Challenges, 2003, Humana Press Inc., Totowa, NJ, 243–266.
72. Zurich, M.G., Honegger, P., Schilter, B., Costa, L.G., Monnet-Tschudi, F., Involvement of glial cells in the neurotoxicity of parathion and chlorpyrifos. Toxicol. Appl. Pharmacol. 201 (2004), 97–104.
73. Zurich, M.G., Stanzel, S., Kopp-Schneider, A., Prieto, P., Honegger, P., Evaluation of aggregating brain cell cultures for the detection of acute organ-specific toxicity. Toxicol. in Vitro 27 (2013), 1416–1424.
74. Zychowicz, M., Dziedzicka, D., Mehn, D., Kozlowska, H., Kinsner-Ovaskainen, A., Stepien, P.P., Rossi, F., Buzanska, L., Developmental stage dependent neural stem cells sensitivity to methylmercury chloride on different biofunctional surfaces. Toxicol. in Vitro 28 (2014), 76–87.