Neuroscience & Medicine, 2012, 3, 321-326
http://dx.doi.org/10.4236/nm.2012.33037 Published Online September 2012 (http://www.SciRP.org/journal/nm) 321
Human Embryo Neuronal Culture in Vitro: A Model to
Study Cellular Physiology, Receptors, Power and Toxicity
of Cytostatic Drugs for Human Use
Stabile Mariano1, Monaco Roberto2, Iuorio Tina1, Buoninfante Luca3, Marino Lucia1,
Altieri Vincenzo4, Della Ragione Carlo2, Masillo Francesco3
1ZigoteGenetic and Prenatal Diagnosis Centre, Salerno, Italy; 2Anatomy Pathology Department, Hospital “A. Cardarelli”, Naples,
Italy; 3Department of Gynecology and Obstetrics, Hospital “Maria SS Addolorata”, Eboli, Italy; 4Genetic Department, Hospital
“Elenad’ Aosta”, Naples, Italy.
Received May 14th, 2012; revised June 14th, 2012; accepted June 22nd, 2012
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 perfo rmed with a rabbit Monoclonal Antibody. ERα was absen t both on neurons grown in vitro an d
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 Sys tem). The absence of nuclear ERα on neurons
in culture and in brain tissue specimens in our expe riment is not in contrast with the relevant physiologic role of estro-
gens on nervous central system, but it could be correlated to the embryonic period of life and could represent a protec-
tion of male brain from an undue estrogens imprinting. The mitomycin C, alkylation agent, has shown in our experi-
ment 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;
Methotrexat e To xi ci t y Test in Vitro
1. Introduction
Neural cells culture has been successfully accomplished
from neural precursors from marine embryonic stem cell
[1] and from human ones [2].
The aim o f the above me ntioned r esearch es was to ob-
tain neural progenitors fit to integrate in vivo into a host
tissue in order to replace damaged or absent neurons
[3-6].
Our in vitro mod el, that is the culture of well differen-
tiated neurons and not stem cells, from human embryo
brain, is important for the research on hormones, drugs,
neuromediators receptors with the aim to understand
their role in morphogenesis and a potential therapeutic
use or neuroto xi c i t y .
In addition, an original con tribution of our paper is the
study of estrogens receptor ERα on neurons in culture
and toxicity test for Mitomycin C and Methotrexate on
neurons in vitro.
2. Materials and Methods
2.1. Embryos Collection
We have obtained the positive judgement by the ethic
committee (Pro t. n . 47 519 /I3D7 A sl Sa 2 the 25 .0 8.2005)
for use of embryonic material from voluntary abortion.
The gestational age of the pregnant women were be-
tween 9˚ - 11˚W; an expert pathologist was in operating
room in order to collect the embryo just delivered, enu-
cleate brain and spinal medulla and, after washing, place
the material in medium culture. Fragmented embryos
were not selected for experiments.
2.2. Neurons in Vitro Culture
Brain and spinal medulla were gently cutted in a Petri
dish, put in a sterile solution of Collagen ase Type I 176.0
units/mg (Gibco) at a concentration of 1 mg/100 ml. Af-
ter 2 hours of incubation at 37˚C in atmosphere of CO2

Human Embryo Neuronal Culture in Vitro: A Model to Study Cellular Physiology, Receptors, Power and
Toxicity of Cytostatic Drugs for Human Use
322
5%, the material was collected in conical tube and cen-
trifuge at 1500 rpm for 10 m’. Thereafter, the sediment
was suspended in culture medium B-27 Electrophysiol-
ogy Kit (Invitrogen) specially formulated to promote an
increased density of synapses and neurotransmitter re-
ceptors; 1 ml/100 of antibiotic antimycotic solution
(100×) (Sigma) was added to the culture medium. The
cellular suspension was plated in glass Petri dishes hav-
ing in the centre a glass slide Superfrost plus (Thermo
Scientific Menzel-Glaser) 25 × 75 × 1 mm, in order to
have a neuronal growth directly on glass slide fit to his-
tochemical analysis; Superfrost plus has an electrostatic-
cally surface which binds better the cells.
Contrast phase microscope observation at 2, 4, 6 and 8
days from setting up the primary culture evidenced the
growth of typically shaped neuronal cells with prolonga-
tions and synapses between cellular processes (Figure
1).
Only three out of the six originally embryo culture
were successfully performed for a period of about 30
days; the other 3 cultures failed because bacterial and
yeast contamination (too high the initial microbial
load).
The embryo sex was established by FISH on inter-
phase neurons culture; the sex was female for all three. In
addition the complete karyotype was successfully ob-
tained through treatment for cytogenetic investigation of
neuron cultures.
2.3. Histochemical Confirmation of Neuronal
Phenotype
High concentration of Neuron Specific Enolase (NSE)
are present in neurons and in neuroendocrine lineage. For
this reason we have used the Mouse Monoclonal Anti-
body anti-NSE (Neuron Specific Enolase-Ventana Medi-
cal System, Inc.) that reacts with NSE localized in the
neuronal cytoplasm cells (Figure 2).
3. Detection of ERα in Neurons in Vitro and
in Brain Specimens
Detection of Estrogen Receptor (ERα) in neuronal cells
was performed with a rabbit Mo noclonal Antibod y (IgG)
(CONFIRM Estrogen Receptor-Ventana Medical System,
Inc.), that is intended for laboratory use for the qualita-
tive detection of ER antigen. It is directed against an
epitope present on human ER protein.
The cells that have in their nucleus the Estrogen Re-
ceptor stain brown and result positive. Absence of nu-
clear histochemical stain of ERα is shown in Figure 3(a)
and absence of immunoreactivity ERα of embryonic
brain specimens fixed in formalin in Figure 3 (b).
(a)
(b)
(c)
(d)
(e)
Figure 1. Neuronal culture: (a) After 2 days; a1 typical neu-
ron; (b) Protoplasmic astrocyte; (c) After 4 days; (d) After 6
days; (e) After 8 days. Neur onal synapses are yet evident by
the 4 days.

Human Embryo Neuronal Culture in Vitro: A Model to Study Cellular Physiology, Receptors, Power and
Toxicity of Cytostatic Drugs for Human Use 323
Figure 2. Immunohistochemical positivity for NSE (Neu-
ronal Specific Enolase) of the neuron grown on glass slide:
staining with anti-NSE antibody is uniformly intense on the
cytosol of the cells confirming the neur onal phenotype.
4. Toxicity Test in Vitro for Two
Antineoplastic Drugs: Mitomycin C and
Methotrexate
The toxicity of the two drugs has been evaluated on two
distinct subcultures in flasks from a primary culture of
embryonic neurons.
Both has been added to the culture medium with a fi-
nal concentration of 50 µg/ml, comparable to blood con-
centrations used in human cancer therapy [7].
After 48 hours from the administration of Mitomycin
C (Mitomycin C 10 mg, Kyowa Pharmaceuticals), the
culture showed clear signs of cellular suffering with most
of the cells in suspension (Figure 4).
Instead, the culture with Methotrexate (Metotrexate 5
mg, Lederle Pharmaceuticals) did not show, after 48
hours, any sign of cytopathy or detachment from the sur-
face monolayer and proliferation was non apparently
inhibited (Figure 5).
(a)
(b)
Figure 3. Histochemical stain of ERα with a rabbit Mono-
clonal Antibody (IgG) (CONFIRM Estrogen Receptor-
Ventana Medical System, Inc.): (a) Absence of brown col-
oration indicating absence of ERα protein, in neurons in
vitro; (b) In brain tissue.
5. Discussion
Human embryonic neurons from spinal cord in culture
have been obtained by Kato A. C. et al. 1985 [8]: bio-
chemical studies demonstrate a prevalence of cholinergic
and GABAergic neurons.
Sah D. W. 1995 [9] has studied voltage and ligand-
gated currents in human foetal central neurons in culture.
Human embryonic neuronal cultures have been estab-
lished in our laboratory without particular difficulty. The
more critical step of the procedure is the collection of
material: an expert operator must be present in the su rgi-
cal room and has to select embryonic material immedi-
ately after the expulsion. A further careful micro-dissect-
tion of the material has been performed in laboratory; it
is better to initiate the cultu re with a redu ced quantity (5 -
10 mg) of embryonic nervous tissue material instead of a
contamination with other cellular type. More than 90 % of

Human Embryo Neuronal Culture in Vitro: A Model to Study Cellular Physiology, Receptors, Power and
Toxicity of Cytostatic Drugs for Human Use
324
Figure 4. Mitomycin C added neuronal culture: reduction
of cellular prolongations and many cells are in suspension
was yet evident at 2 days from subc ultur es.
Figure 5. Me totrexate added neuronal culture: no cytotoxic
effect of metotrexate was apparent at 2, 4, 6 days from
subculture.
cells in vitro in our experiment are neurons, astrocytes,
oligodendrocytes and Neural Stem Cell on the basis of
morphological aspect and histochemical study.
A protective rule of estrogens in neurodegenerative
diseases has been postulated [10,11].
The two types of Estrogens Receptors (ERs), ERα and
ERβ, belong to the nuclear receptor superfamily, a family
of ligand-regulated transcription factor. Both receptors
are coexpressed in a number of tissue and form homo-
dimers (α/α, β/β) and heterodimers (α/β) [12]. When co-
expressed, ERβ inhibits the ERα mediated gene expres-
sion. In mice ERα is expressed primarily in the uterus,
liver, kidney, and heart, whereas ERβ is expressed pri-
marily in the ovary, prostate, lung, gastrointestinal tract,
bladder, hematopoietic and central nervous systems
[13-15]. Cells containing immunoreactive estrogen re-
ceptor-alpha have been found in the human basal fore-
brain [16,17]. Differential Expression of Estrogen Re-
ceptor α and β immunoreactivity has been found in the
Human Supraoptic Nucleus (dl-SON) in Relation to Sex
and Aging [18]. Significant correlations between the
percentage of ERβ- and ERα-positive and -negative AVP
(Plasma Arginine Vasopressin) neurons and age were
found in women, but not in men: a strong decrease of ER
β and an in crease of ERα immunoreactivity in AVP neu-
rons of the dl-SON of postmenopausal women. Both re-
ceptor changes could participate in the activation of the
AVP neurons in postmenopausal wome n.
Estrogen receptors localization in the human spinal
trigeminal nucleus has been evidenced by Fenzi e
Rizzuto [19].
Although ER subtypes may be expressed in the same
tissue, they may not be expressed in the same cell type.
Nonetheless, ERα and ERβ proteins have been simulta-
neously detected in many cell types including neurons.
The absence of nuclear ERα on neurons in culture and in
brain tissue specimens in our experiment is not in con-
trast 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. A role
of progesterone in inducing human embryonic stem cell
proliferation and differentiation into neuroectodermal
rosettes has been established [20].Considering that the
two hormones, estrogens and progesterone, often have an
antagonistic effect, the absence of ERα receptor in em-
bryonic brain could have its significance in non-antago-
nize the morphogenetic effect of progesterone.
Mitomycin C and metotrexate are antineoplastic drugs
present in many polichemiotherapic schedules, including
brain cancer.
The mitomycin C, alkylating agent, has shown in our
experiment a major neurotoxic and cytostatic power in
comparison with methotrexate.
Metotrexate, competitive antagonist of the folic acid
on catalytic site of the dihydrofolate reductase (DHFR),
has an action which can be weakened by three factors:
the reduction of polyglutamate inside the cell, increase of
DHFR by genetic amplification, reduction of intracellular
transport [7]. Therefore the apparent absence of neuro-
toxic effect in our model need a more complex experi-
mental design to be explained. Nevertheless, the thera-
peutic index of Metotrexate is better than Mitomycin C
on the basis of our experi mental findi n g.
Figure 6. Metaphase with GTL banding from human neu-
rons in culture.

Human Embryo Neuronal Culture in Vitro: A Model to Study Cellular Physiology, Receptors, Power and
Toxicity of Cytostatic Drugs for Human Use 325
The cytogenetic investigation has easily been per-
formed on our human embryonic neurons (Figure 6); the
analysis of chromosomal fragility and SCE (Sister
Chromatide Exchange) is another powerful method to
test the clastogenic activity of many substances for hu-
man therapy [21,22].
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 Neurosci-
ence, Vol. 10, 2009, p. 122.
doi:10.1186/1471-2202-10-122
[2] M. Schuldiner, O. Yanuka, J. Itskovitz-Eldor, D. A. Mel-
ton and N. Benvenisty, “Effects of Eight Growth Factors
on the Differentiation of Cells Derived from Human Em-
bryonic Stem Cells,” Proceedings of the National Acad-
emy 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. Mack-
lis, “Adult Neurogenesis and Cellular Brain Repair with
Neural Progenitors, Precursors and Stem Cells,” Phi-
losophical Transactions of the Royal Society B: Biologi-
cal 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 Differentia-
tion 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. Sundström, “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 Spinocerebellara-
taxia 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 Electro- phy-
siological Properties,” The Journal of Neuroscience Co-
pyright 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 Neuro-
physiology, 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 Estro-
gens (Premarin®) Are Protective against Neurodegenera-
tive Insults: Implications for a Composition of Estrogen
Therapy to Promote Neuronal Function and Prevent Alz-
heimer’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,” Pharma-
cological Reviews, Vol. 62, No. 2, 2010, pp. 155-198.
doi:10.1124/pr.109.002071
[12] J. Matthews and J. A. Gustafsson, “Estrogen Signaling: A
Subtle Balance between ER Alpha and ER Beta,” Mo-
lecular 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 Sero-
tonin 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 Func-
tion,” 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, “Oes-
trogen Receptor Alpha Localisation in the Prefrontal
Cortex of Three Mammalian Species,”Journal of Neuro-
endocrinology, 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 Supra-
optic 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. Bo-
wen, S. V. Meethal and C. S. Atwood, “The Pregnancy
Hormones Human Chorionic Gonadotropin and Proges-
terone Induce Human Embryonic Stem Cell Proliferation
and Differentiation into Neuroectodermal Rosettes,” Stem

Human Embryo Neuronal Culture in Vitro: A Model to Study Cellular Physiology, Receptors, Power and
Toxicity of Cytostatic Drugs for Human Use
326
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 In-
creased Risk for Cancer,” Environmental Health Perspec-
tives, 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