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

doi:10.4236/nm.2012.33037

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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.

Email: [email protected]

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].

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