Human Embryo Neuronal Culture in Vitro: A Model to Study Cellular Physiology, Receptors, Power and Toxicity of Cytostatic Drugs for Human Use

Stabile Mariano, Monaco Roberto, Iuorio Tina, Buoninfante Luca, Marino Lucia, Altieri Vincenzo, Della Ragione Carlo, Masillo Francesco

Anatomy Pathology Department, Hospital “A. Cardarelli”, Naples, Italy.
Department of Gynecology and Obstetrics, Hospital “Maria SS Addolorata”, Eboli, Italy.
Genetic Department, Hospital “Ele-nad’ Aosta”, Naples, Italy.
ZigoteGenetic and Prenatal Diagnosis Centre, Salerno, Italy.

DOI: 10.4236/nm.2012.33037                

Abstract

Neural cells cultures from human embryo brain of 9° - 11°W gestational age have been used to study ERα (Estrogens Receptor α) and to perform toxicity test for Mitomycin C and Methotrexate. Histochemical confirmation of cellular neuronal phenotype was based on histochemical evidence of NSE (Neuron Specific Enolase).The detection of ERα in neuronal cells was performed with a rabbit Monoclonal Antibody. ERα was absent both on neurons grown in vitro and on tissue brain specimens. This finding is apparently in contrast with the positive immunoreactivity of ERα and ERβ reported by other Authors on foetal and adult CNS (Central Nervous System). The absence of nuclear ERα on neurons in culture and in brain tissue specimens in our experiment is not in contrast with the relevant physiologic role of estrogens on nervous central system, but it could be correlated to the embryonic period of life and could represent a protection of male brain from an undue estrogens imprinting. The mitomycin C, alkylation agent, has shown in our experiment a major neurotoxic and cytostatic power in comparison with methotrexate. Our conclusion is that human embryo neuronal culture in vitro is a powerful instrument for physiology and human therapy for cancer and neurodegenerative diseases.

Keywords

Human Embryo Neuronal Culture; ERα on Embryonic Brain; Mitomycin C Toxicity Test in Vitro; Methotrexate Toxicity Test in Vitro

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	H. W. Li, H. Liu, C. E. Corrales, J. R. Risner, J. Forrester, J. R. Holt, S. Heller and A. S. B. Edge, “Differentiation of neurons from Neural Precursors Generated in Floating Spheres from Embryonic Stem Cells,” BMC Neuroscience, Vol. 10, 2009, p. 122. doi:10.1186/1471-2202-10-122
[2]	M. Schuldiner, O. Yanuka, J. Itskovitz-Eldor, D. A. Melton and N. Benvenisty, “Effects of Eight Growth Factors on the Differentiation of Cells Derived from Human Embryonic Stem Cells,” Proceedings of the National Academy of Sciences of USA, Vol. 97, No. 21, 2000, pp. 11307- 11312. doi:10.1073/pnas.97.21.11307
[3]	U. S. Sohur, J. G. Emsley, B. D. Mitchell and J. D. Macklis, “Adult Neurogenesis and Cellular Brain Repair with Neural Progenitors, Precursors and Stem Cells,” Philosophical Transactions of the Royal Society B: Biological Sciences, Vol. 361, No. 1473, 2006, pp. 1477-1497. doi:10.1098/rstb.2006.1887
[4]	C. E. Corrales, L. Pan, H. W. Li, M. C. Liberman, S. Heller and A. S. B. Edge, “Engraftment and Differentiation of Embryonic Stem Cell-Derived Neural Progenitor Cells in the Cochlear Nerve Trunk: Growth of Processes into the Organ of Corti,” Journal of Neurobiology, Vol. 66, No. 13, 2006, pp. 1489-1500. doi:10.1002/neu.20310
[5]	M. Emgard, L. Holmberg, E. B. Samuelsson, B. A. Bahr, S. Falci, A. Seiger and E. Sundstr?m, “Human Neural Precursor Cells Continue to Proliferate and Exhibit Low Cell Death after Transplantation to the Injured Rat Spinal Cord,” Brain Research, Vol. 1278, 2009, pp. 15-26. doi:10.1016/j.brainres.2009.04.012
[6]	Y.-K. Chang, M.-H. Chen, Y.-H. Chiang, Y.-F. Chen, W.-H. Ma, C.-Y. Tseng, B.-W. Soong, J. H. Ho and O. K. Lee, “Mesenchymal Stem Cell Transplantation Amelio-ratesmotor Function Deterioration of Spinocerebellarataxia by Rescuing Cerebellar Purkinje Cells,” Journal of Biomedical Science, Vol. 18, 2011, p. 54 doi:10.1186/1423-0127-18-54
[7]	E. Chu and A. Sartorelli, “Antineoplastic Chemotherapy,” In: Katzung Basic & Clinical Pharmacology, 9th Edition, The McGraw-Hill Companies, Inc., 2004, pp. 898-932
[8]	A. C. Kato, G. Touzeau, D. Bertrand and C. R. Bader, “Human Spinal Cord Neurons in Dissociated Monolayer Cultures: Morphological, Biochemical, and Electrophysiological Properties,” The Journal of Neuroscience Copyright Society for Neuroscience, Vol. 5, No. 10, 1985, pp. 2750-2761.
[9]	D. W. Sah, “Human Fetal Central Neurons in Culture: Voltage- and Ligand-Gated Currents,” Journal of Neurophysiology, Vol. 74, No. 5, 1995, pp. 1889-1899.
[10]	L. Q. Zhao and R. D. Brinton, “Select Estrogens within the Complex Formulation of Conjugated Equine Estrogens (Premarin?) Are Protective against Neurodegenerative Insults: Implications for a Composition of Estrogen Therapy to Promote Neuronal Function and Prevent Alzheimer’s Disease,” BMC Neuroscience, Vol. 7, 2006, p. 24. doi:10.1186/1471-2202-7-24
[11]	G. E. Gillies and S. McArthur, “Estrogen Actions in the Brain and the Basis for Differential Action in Men and Women: A Case for Sex-Specific Medicines,” Pharmacological Reviews, Vol. 62, No. 2, 2010, pp. 155-198. doi:10.1124/pr.109.002071
[12]	J. Matthews, J. A. Gustafsson, “Estrogen Signaling: A Subtle Balance between ER Alpha and ER Beta,” Molecular Interventions, Vol. 3, No. 5, 2003, pp. 281-292.
[13]	Z. Sheng, J. Kawano, A. Yanai, R. Fujinaga, M. Tanaka, Y. Watanabe and K. Shinoda, “Expression of Estrogen Receptors (Alpha, Beta) and Androgen Receptor in Serotonin Neurons of the Rat and Mouse Dorsal Raphe Nuclei; Sex and Species Differences,” Neuroscience Research, Vol. 49, No. 2, 2004, pp. 185-196.
[14]	R. J. Handa, S. Ogawa, J. M. Wang and A. E. Herbison, “Roles for Estrogen Receptor Beta in Adult Brain Function,” Journal of Neurobiology, Vol. 24, 2011, pp. 160-173.
[15]	B. Greco, E. A. Allegretto, M. J. Tetel and J. D. Blaustein, “Coexpression of ER_ with ER and Progestin Receptor Proteins in the Female Rat Forebrain: Effects of Estradiol Treatment,” Endocrinology, Vol. 142, 2001, pp. 5172- 5181. doi:10.1210/en.142.12.5172
[16]	J. E. Donahue, E. G. Stopa, R. L. Chorsky, J. C. King, H. M. Schipper, S. A. Tobet, J. D. Blaustein and S. Reichlin, “Cells Containing Immunoreactive Estrogen Receptor-Alpha in the Human Basal Forebrain,” Brain Researvh, Vol. 856, No. 1-2, 2000, pp. 142-151. doi:10.1016/S0006-8993(99)02413-0
[17]	D. Montague, C. S. Weickert, E. Tomaskovic-Crook, D. A. Rothmond, J. E. Kleinman and D. R. Rubinow, “Oestrogen Receptor Alpha Localisation in the Prefrontal Cortex of Three Mammalian Species,”Journal of Neuroendocrinology, Vol. 20, No. 7, 2008, pp. 893-903. doi:10.1111/j.1365-2826.2008.01743.x
[18]	T. A. Ishunina, F. P. Kruijver, R. Balesar and D. F. Swaab, “Differential Expression of Estrogen Receptor Alpha and Beta Immunoreactivity in the Human Supraoptic Nucleus in Relation to Sex and Aging,” Journal of Clinical Endocrinology and Metabolism, Vol. 85, No. 9, 2000, pp. 3283-3291. doi:10.1210/jc.85.9.3283
[19]	F. F. N. Rizzzuto, “Estrogen Receptors Localization in the Spinal Trigeminal Nucleus: An Immunohistochemical Study in Humans,” European Journal of Pain, Vol. 15, No. 10, 2011, pp. 1002-1007.
[20]	M. J. Gallego, P. Porayette, M. M. Kaltcheva, R. L. Bowen, S. V. Meethal and C. S. Atwood, “The Pregnancy Hormones Human Chorionic Gonadotropin and Progesterone Induce Human Embryonic Stem Cell Proliferation and Differentiation into Neuroectodermal Rosettes,” Stem Cell Research & Therapy, Vol. 1, 2010, p. 28.
[21]	D. Jacobson-Kram, R. J. Albertini, R. F. Branda, M. T. Falta, P. T. Iype, K. Kolodner, S.-H. Liou, M. A. Mc-Diarmid, M. Morris, J. A. Nicklas, J. P. O’Neill, M. C. Poirier, D. Putman, P. T. Strickland, J. R. Williams and S. Xiao, “Measurement of Chromosomal Aberrations, Sister Chromatid Exchange, Hprt Mutations, and DNA Adducts in Peripheral Lymphocytes of Human Populations at Increased Risk for Cancer,” Environmental Health Perspectives, Vol. 101, No. 3, 1993, pp. 121-125.
[22]	K. Siviková, J. Dianovsky and B. Holecková, “Induction of SCEs and DNA Fragmentation in Bovine Peripheral Lymphocytes by in vitro Exposure to Tolylfluanid-Based Fungicide,” Genetics and Molecular Biology, Vol. 34, No. 1, 2011, pp. 110-115. doi:10.1590/S1415-47572010005000097