Neural stem cells are a potential transplantation source for ...

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Jinhao Sun 1 ,LinYang 1 , Zhenhua Li 1 ,JiazengWang 1 ,FulingCao 2 ,LihuaBao 1 , Wenjing Liu 1 ,,ZhiyuLiu 1
1.Department of Anatomy, School of medicine, Shandong University, Jinan, 250012, China
2.Department of Surgery, Yicheng Regional Hospital of Traditional Chinese Medicine
To evaluate the effects of epithelial growth factor (EGF) on the growth and differentiation of
embryonic neuroepithelial stem cells. Neuroepithelial stem cells were dissociated from the neural tube of
embryonic day 11(E11) rats and cultured with EGF and control medium. Micromeasure technique and immunocy 鄄
tochemical staining were employed to detect neurite growth and expression of neuron-specific enolase (NSE) and
glial fibrillary acidic protein (GFAP) of neuroepithelial stem cells. The neurite length in EGF groups was
longer than that in control group at 24h 尧 48h and 72h after culture. Immunohistochemical staining showed that
more NSE-positive and GFAP-positive cells were found in EGF groups.
EGF could promote the
growth and differentiation of neural tube-derived neuroepithelial stem cells.
Neuroepithelial stem cells; Differentiation; EGF
Neural stem cells are a potential transplantation
source for neurodegenerative disease. It is known
that these cells have the ability to differentiate into
different kinds of neurons and glial cells. Many
growth factors, like EGF, can regulate the expansion
and differentiation of neural stem cells. As an indis 鄄
pensable growth factor, EGF may promote neural stem
cells proliferation in serum free culture medium. Fur 鄄
thermore, EGF is capable to induce the differentiation
of neural stem cells derived from embryonic cortex,
embryonic cerebellum, and from adult brain, adult
spinal cord, et al [1-3] .
During the process of development, neural plate
folds into the cylindrical neural tube, which is the em 鄄
bryonic structure that develops into the brain and
spinal cord. Neuroepithelial stem cells in neural tube
are the most primitive neural stem cells which can
form the whole central nervous system [4] . The expres 鄄
sion of EGF and EGF receptor was high in neuroep 鄄
ithelial stem cells [5] . However, to this day, there have
Correspondence author: Lin Yang, Professor,
Email: yanglin@sdu.edu.cn
*This program was supported by three grants:
Shandong research development program (No.031050144),
Shandong province excellent young scientist program (No.
2004BS02012) and Shandong Hygiene Department young
scholar program.
been very limited studies on the effects of EGF on
these primitive cells. Here, we give a study in that di 鄄
rection.
DMEM/F12 (Gibco), EGF (sigma), new calf serum
(Hangzhou Sijiqing Company), NSE and GFAP anti 鄄
bodies were bought from Beijing Zhongshan Biologi 鄄
cal Product Company. Wistar Pregnant rats were used
in experiment.
Neural tube was obtained from 11 days embryo
(post-conception) under compliance with National In 鄄
stitute of Health Guidelines. The tissue of neural tube
was dissected in sterile saline, and neuroepithelial stem
cells were isolated. Then, cells were cultured in
DMEM/F12 medium supplemented with 10% heat-in 鄄
activated new calf serum, streptomycin 100mg.L -1 ,
benzylpenicilin 100kU.L -1 ,ina5%CO 2 humidified in 鄄
cubator at 37 益 , at a density of 100,000cells/ml. The
cultured cells were divided into two groups: the medi 鄄
um of experiment group (n=6) containing EGF 60ng/
ml, and control group (n =6) only containing normal
medium.

44
Jinhao Sun: Effect Of Epithelial Growth Factor On Growth......
EGF promotes neurite growth of neuroepithelial stem cell
Embryonic neural stem cells differentiate into NSE-posi 鄄
tive cells at 7 days cultured with EGF. (SABC 伊 200)
Under microscope, the length of neurite was exam 鄄
ined after 8h, 24h, 48h and 72h in vitro culture. 10 vi 鄄
sual fields (200 伊 ) were selected randomly both in ex 鄄
periment group and control group and the neurite
lengths of every cell in the visual fields were mea 鄄
sured. Among many neurite of cell body, the longest
one was observed.
At 7 days in vitro culture, cells were fixed in 4%
paraformaldehyde in PBS for 10 minutes at room tem 鄄
perature, then pretreated with 50% methanol and 3%
hydrogen peroxide in PBS for 20 min, washed three
times in PBS, and incubated in 10% normal goat
serum (NGS) in PBS for 60 min prior to overnight in 鄄
cubation on a shaking platform with the primary anti 鄄
body (NSE 1:200, or GFAP 1:200). After a 10 -min
rinse in PBS and two 10 -min washes in 5% NGS,
sections were incubated in biotinylated secondary anti 鄄
body (goat-anti-rabbit) at a dilution of 1:200 in 2%
NGS in PBS at room temperature for 60 耀 90min. The
cells were then rinsed three times in PBS and incubat 鄄
ed in avidin-biotin complex (vectastain ABC kit.) in
PBS for 60 耀 90 min at room temperature. Following
thorough rinsing with PBS and Tris -buffered saline,
cultures were developed for 5 耀 10 min in 0.04% hy 鄄
drogen peroxide and 0.05% 3,3 -diaminobenzidine
(sigma) in Tris-buffered saline. Controls with omis 鄄
sion of the primary antibody were performed to verify
the specificity of staining.
Embryonic neural stem cells differentiate into GFAP-posi 鄄
tive cells at 7 days cultured with EGF. (SABC 伊 200)
The length of cell neurite was presented as
meanSEM. A student t -test was used for statistical
analysis.
Neuroepithelial stem cells were cultured in the
presence of EGF or normal medium. In these cultures,
EGF may promote more neurite growth than control.
Furthermore, the neurite length of EGF groups was all
higher than that in control at 24h, 48h and 72h in vitro

Jinhao Sun: Effect Of Epithelial Growth Factor On Growth...... 45
culture (all 0.05) ,see Fig.1.
Immunochemical staining was done to assess the
expression of NSE and GFAP, which were the markers
of neuron and astrocyte respectively. At 7 days in vit 鄄
ro culture, more NSE-positive cells were detected in
EGF groups than that in control groups (Fig.2), with
many neurite protruded from cell body. Although
GFAP-positive cells could be detected in both groups,
there were more GFAP-positive cells in the presence
of EGF in culture (Fig.3).
Growth factor BDNF is known to promote neuronal
survival and differentiation [9] and its role in neural
stem cell development need to be clarified.
Neural tube is the tissue that will develop into
brain and spinal cord. And, Neuroepithelial stem cells
are the main component of early neural tube. Besides,
these cells keep lively ability to proliferation, and, also
can differentiate into all kinds of neurons and glial
cells in central nervous system. Thus, these cells may
be a potential transplantation source for neurodegener 鄄
[10]
ative disease . More studies should be focused on
these cells.
In summary, we conclude that EGF could promote
growth and differentiation of neuroepithelial stem cells
in neural tube, Here, we only give a very primitive ex 鄄
periment, the mechanisms should be studied further.
Our result indicated that EGF indeed could pro 鄄
mote the neurite growth of neuroepithelial stem cell.
In the presence of EGF, cell bodies protrude longer
neurite than that in control group. Further, we detected
more NSE-positive cells and GFAP-positive cells in
EGF group. This denotes that a high percentage neu 鄄
rons and glial cells have differentiated from the em 鄄
bryonic neuroepithelial stem cells. In fact, during early
embryo development, expressions of both EGF and
EGFR are high in neural tube, which suggests that
EGF may play an important role in neural develop 鄄
ment [6] . Our results provide this evidence that EGF in 鄄
deed promotes growth and differentiation of early em 鄄
bryonic neuroepithelial stem cells in neural tube. Some
similar reports said that EGF might stimulate differen 鄄
tiation of neural stem cells derived from other regions
[7].
Except for EGF, many other growth factors also
have the ability to promote the growth and differentia 鄄
tion of neural stem cells , such as basic fibroblast
growth factor 渊 bFGF 冤 , brain derived neurotrophic fac 鄄
tor, etc. Kelly and his colleagues reported that bFGF 鄄
may induce the proliferation of embryonic neural pre 鄄
cursors in vitro. And, they also demonstrate that em 鄄
bryonic age is a crucial determinant of the number and
differentiation potential of rat embryonic neural pre 鄄
cursor cells responding to either EGF and/or bFGF [8] .
1. Gage FH, Ray J, Fisher LJ. Isolation, characterization, and
use of stem cells from the CNS. Annu Rev Neurosci, 1995,
18:159-192.
2. Galvin KA and Jones DG. Adult human neural stem cells
for cell-replacement therapies in the central nervous system.
Med J Aust, 2002, 177 :316-318.
3. Represa A, Shimazaki T, Simmonds M, Weiss S. EGF-re 鄄
sponsive neural stem cells are a transient population in the
developing mouse spinal cord. Eur J Neurosci, 2001, 14:452-
62.
4.ZhangX,KlueberKM,GuoZ,CaiJ,LuC,WinsteadWI,
Qiu M, Roisen FJ. Induction of neuronal differentiation of
adult human olfactory neuroepithelial -derived progenitors.
Brain Res, 2006, 16: 109-119.
5. Weston CR, Wong A, Hall JP, Goad ME, Flavell RA,
Davis RJ. The c -Jun NH2 -terminal kinase is essential for
epidermal growth factor expression during epidermal morpho 鄄
genesis. Proc Natl Acad Sci U S A, 2004, 101: 14114-14119.
6. Boockvar JA, Kapitonov D, Kapoor G, Schouten J,
Counelis GJ, Bogler O,Snyder EY, McIntosh TK, O' Rourke
DM. Constitutive EGFR signaling confers a motile phenotype
to neural stem cells. Mol Cell Neurosci, 2003, 24:1116-1130.
7. Kelly CM, Tyers P, Borg MT, Svendsen CN, Dunnett SB,
Rosser AE. EGF and FGF-2 responsiveness of rat and mouse
neural precursors derived from the embryonic CNS. Brain Res
Bull, 2005, 68:83-94.
8. Kelly CM, Tyers P, Borg MT, Svendsen CN, Dunnett SB,
Rosser AE.EGF and FGF-2 responsiveness of rat and mouse

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Jinhao Sun: Effect Of Epithelial Growth Factor On Growth......
neural precursors derived from the embryonic CNS. Brain Res
Bull, 2005,68:83-94.
9. Cheng A, Wang S, Cai J, Rao MS, Mattson MP.Nitric ox 鄄
ide acts in a positive feedback loop with BDNF to regulate
neural progenitor cell proliferation and differentiation in the
mammalian brain. Dev Biol, 2003, 258:319-333.
10. Rafael H. Neural transplantation. J Neurosurg, 2006, 104:
336-337.
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