MINISTRY OF EDUCATION & TRAINING
CAN THO UNIVERSITY
BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE
SUMMARY
BACHELOR OF SCIENCE THESIS
THE ADVANCED PROGRAM IN BIOTECHNOLOGY
EVALUATION EFFECTS OF LINGZHI
MUSHROOM (Ganoderma lucidum) ON NEURAL
STEM CELLS ISOLATED FROM EMBRYONIC
MOUSE BRAIN (Mus musculus var. albino)
SUPERVISORS
STUDENT
Dr. PHAM VAN PHUC
NGUYEN THI MAI DAN
MSc. TRUONG HAI NHUNG
Student ID code: 3082584
Session: 34 (2008- 2013)
Ho Chi Minh City, 05/2013
APPROVAL
SUPERVISORS
Dr. PHAM VAN PHUC
STUDENT
NGUYEN THI MAI DAN
MSc. TRUONG HAI NHUNG
Can Tho,
PRESIDENT OF EXAMINATION COMMITTEE
ABSTRACT
Neurodegenerative diseases or neurological damages such
as Parkinson, Alzheimer, Huntington and spinal cord injury have
become a great challenge to public health. In fact, treatment for
these diseases is restricted since new neural cells cannot be
generated by the nervous system, thus an efficient therapy is
emergently required.
Recently, stem cells, especially neural stem cells (NSCs),
are employed as an innovative tool for the replacement of
damaged cells due to self-renewal as well as pluripotent capacity.
NSCs were isolated from 13.5 – 15.5 post coitum embryonic
mouse brain and cultured in DMEM/F12 serum-free medium
modified with B27, N2, heparin, EGF and FGF at 37o C, 5% CO2.
Candidate
cells
were
identified
for
neural
stem
cell
characteristics by neurosphere assay, the expression of Nestin,
CD133, Sox-1 and GFAP. Those NSCs were then cultured in 96well plate added with Ganoderma lucidum extract at 100µg/ml,
500µg/ml, 1000µg/ml for the evaluation of medicinal effects of
such mushroom on cell proliferation. The results revealed that
72% of sample was successfully cultured, cells isolated from
13.5-15.5 day post coitum fetus showed the best capacity of
culture, isolated cells could formed neurosphere, expressed Sox1,
CD133, Nestin and GFAP. Extract at 500µg/ml showed the best
effects on neural stem cell growth.
Keywords: Ganoderma lucidum, neurosphere assay, neural
stem cell markers, neurosphere size.
i
CONTENTS
ABSTRACT ...............................................................................i
CONTENTS ..............................................................................ii
CHAPTER 1: INTRODUCTION ............................................. 1
CHAPTER 2: MATERIALS AND METHODS ..................... 3
2.1 Materials ............................................................................... 3
2.2 Methods ................................................................................ 5
2.2.1 Activity 1: Isolation and culture of candidate NSCs
isolated from embryonic mouse brain (Mus
musculus var. albino)...................................................... 5
2.2.1.1 Isolation and primary culture of cells isolated
from embryonic mouse brain .................................... 5
2.2.1.2 Secondary culture of candidate NSCs ........................ 5
2.2.2 Activity 2: Determination of characteristics of
isolated candidate cells.................................................. 6
2.2.2.1 Neurosphere assay .................................................... 6
2.2.2.2 Expression of markers of candidate NSCs ................. 6
2.2.2.2.1 Determination of Sox1 expression of
candidate NSCs by RT-PCR (reverse
transcription polymerase chain reaction) ......... 6
2.2.2.2.2 Determination of CD133 expression of
candidate NSCs by flow cytometry ................. 7
2.2.2.2.3 Determination of Nestin expression of
candidate
NSCs
by
immuno
-
cytochemistry ................................................. 7
2.2.2.3 Differentiation to astrocytes and GFAP expression.... 8
ii
2.2.3 Activity 3: Evaluation effects of Ganoderma
lucidum on cell proliferation ......................................... 9
2.2.4 Data analysis ................................................................... 9
CHAPTER 3: RESULTS AND DISCUSSION ...................... 10
3.1 Isolation and culture of candidate NSCs from
embryonic mouse brain ..................................................... 10
3.1.1 Isolation and primary culture of candidate NSCs............ 10
3.1.2 Secondary culture .......................................................... 11
3.2 Determination of characteristics of candidate NSCs ............. 12
3.2.1 Neurosphere assay ......................................................... 12
3.2.2 Expression of markers of candidate NSCs...................... 13
3.2.2.1 Sox1 expression of candidate NSCs......................... 13
3.2.2.2 CD133 expression of candidate NSCs ..................... 14
3.2.2.3 Nestin expression of candidate NSCs ...................... 15
3.2.2.4 Differentiation and GFAP expression of
candidate NSCs ...................................................... 16
3.3. Effects of Ganoderma lucidum on neural stem cell
proliferation ...................................................................... 17
CHAPTER 4: CONCLUSION AND SUGGESTION ............ 20
4.1 Conclusion .......................................................................... 20
4.1.1 Isolation and culture of NSCs ........................................ 20
4.1.2 Neural stem cell characteristics determination ................ 20
4.1.3 Cell proliferation effects of Ganoderma lucidum
extract .......................................................................... 20
4.2 Suggestion .......................................................................... 20
REFERENCES ....................................................................... 22
iii
CHAPTER 1: INTRODUCTION
Neurodegenerative diseases, the quantity loss of functional
neural cells in the brain, spinal cord and nerves due to apoptosis
or necrosis, could affect body movement, understanding,
respiration, emotion and memory.
According to WHO (World Health Organization), there
were 1 billion patients suffering neurodegenerative diseases in
2007, with 50 million cases of epilepsy and 24 million cases of
Alzheimer, resulting in 6.8 million cases of death annually. Thus,
neurodegenerative diseases, with their dramatic growth, have
been raising a great concern among general public over the past
65 years. Among these, Alzheimer’s disease and Parkinson’s
disease are the most popular neurological disorders in the world.
Such diseases have been treated by medication or conventional
surgery; however, many side-effects of medicine were addressed.
In the same context, surgery was reported as an inefficient
therapy since patients had to undergo surgery for several times,
but some symptoms still remained.
Recent studies have discovered potential uses of stem cells
in treating such disorders, especially Parkinson’s disease due to
its enormous efficacy. However, stem cell population is limited in
mature tissues, thus embryonic stem cells with higher selfrenewal and pluripotent capacity are employed. Besides, neural
stem cells (NSCs) from endogenous sources do not efficiently
migrate to specific regions in brain for replacement demand. As a
consequence, extrinsic factors are used to trigger appropriate
signals for cell proliferation and differentiation (Reimers, 2008).
1
The study “Evaluation effects of Ganoderma lucidum on neural
stem cells isolated from embryonic mouse brain (Mus musculus
var. albino)” was conducted to clarify medicinal effects of such
mushroom on cell proliferation at in vitro level and set up a
foundation for further research.
Objective:
- Isolating and culturing neural stem cells from embryonic mouse
brain.
- Determining the best concentration of Ganoderma lucidum for
neural stem cell growth in vitro.
2
CHAPTER 2: MATERIALS AND METHODS
2.1 Materials
Period: December, 2012 – May, 2013
Location: Stem Cell Research and Application Laboratory,
Ho Chi Minh University of Science, Vietnam National
University.
Sample: cells from embryonic mouse brain (Mus musculus
var. albino). 13.5-15.5 day post coitum mouse were purchased
from Pasteur Institute, Ho Chi Minh City.
Ganoderma lucidum extract was purchased from National
Institute of Medicinal Materials, Ho Chi Minh City.
Chemicals:
- Phosphate buffered saline (-)
- PBS modified with Streptomycin and Penicillin (1X, 5X, 10X)
- Trypsin/EDTA solution
- RT-PCR chemicals: Primers (Sox1, GAPDH), One-step RTPCR pre-mix (iNtRON Biotechnology)
- Electrophoresis chemicals: 1.5% agarose gel, 0.5X TAE buffer
- Immunocytochemistry chemicals:
+ Anti-Nestin antibody conjugated with FITC
+ Anti-CD133 antibody conjugated with FITC
+ Anti-GFAP antibody conjugated with Rhodamine
+ Hoechst 33342
- Flow cytometry chemical: FACS Fluid
3
- NSC medium
Chemicals
Concentration
H-DMEM/F12
94.3ml/l
30% Glucose
2 ml/l
Hepes buffer 1M
0.5 ml/l
Progesterone 1000X
0.1 ml/l
Putrescine 1000X
1 ml/l
EGF
20 ml/l
FGF
20 ml/l
Antibiotics
1 ml/l
N2, B27 growth supplement (Invitrogen, USA)
- Differentiation medium
Chemicals
Concentration
H-DMEM/F12
89ml/l
Heparin
25µg/ml
Insulin
5µg/ml
Transferrin
5µg/ml
Gentamycine
1ml/l
N2, B27 growth supplement (Invitrogen, USA)
4
2.2 Methods
2.2.1 Activity 1: Isolation and culture of candidate NSCs
isolated from embryonic mouse brain (Mus musculus var.
albino)
2.2.1.1 Isolation and primary culture of cells isolated from
embryonic mouse brain
NSCs were collected from 13.5-15.5 day post coitum fetus
by abdominal surgery and kept in 10X PBS solution.
Mouse fetus was decontaminated in 10X, 5X and 1X PBS
solution, respectively. Those fetuses were then decapitated and
washed by 1X PBS before brain isolation. Isolated mouse brain
was mechanically dissected, suspended and filtered through a
70µm cell strainer. The dissociated cells were collected by
centrifugation at 2500rpm for 5 minutes and were resuspended in
1ml free-serum NSC medium. Such cells were cultured in 6-well
plates and incubated at 37oC and 5% CO2.
2.2.1.2 Secondary culture of candidate NSCs
Since living cells consumed nutrients, increased in size and
produced waste, which could be toxic to themselves, during
proliferation; thus, cell passages were taken to remove toxic
substances, provide nutritious elements and space for cell growth.
After 2-3 days of culture, suspended neurospheres were
collected by centrifugation at 1500rpm for 5 minutes,
mechanically dissociated and resuspended in 2ml NSC medium,
followed by being subcultured in new wells at 37oC, 5% CO2.
5
2.2.2 Activity 2: Determination of characteristics of candidate
NSCs
2.2.2.1 Neurosphere assay
Neurospheres of passage 4 were collected by centrifugation
at 1500rpm for 5 minutes, resuspended in 2ml NSC medium and
mechanically dissociated. These single cells were then counted by
NucleoCounter and plated at a density of 104 cells/ml in 96-well
plate at 37oC, 5% CO2. Cell proliferation was observed and
evaluated under inverted optical microscope every 24 hours.
2.2.2.2 Expression of markers of candidate NSCs
2.2.2.2.1 Determination of Sox1 expression of candidate NSCs
by RT-PCR (reverse transcription polymerase chain reaction)
For RT-PCR analysis, spheres of passage 4 were collected
by centrifugation at 1500rpm for 5 minutes prior to RNA
extraction. RNA was then extracted by using easy-BLUE kit
(iNtRON Biotechnology) and RT-PCR was performed with Onestep RT-PCR premix kit (iNtRON Biotechnology). Expression
level of Sox1 was measured by electrophoresis on 1.5% agarose
gel (110V, 30 minutes).
Table 3.1 RT-PCR mix
No.
Components
Concentration
Volume
1
RNA
500ng/µl
1.0µl
2
H2O
-
5.5µl
3
One-step RT-PCR pre-mix
-
7.5µl
4
Primers (forward and reverse)
0.25mM
1.0µl
6
70oC
42oC
60
mins
5
mins
95oC
95oC
10
mins
15
mins
toC
gradient
30 secs
72o
C
30
secs
72o
C
10
mins
4oC
∞
30 cylces
Figure 3.4 Thermal cycle of RT-PCR
2.2.2.2.2 Determination of CD133 expression of candidate
NSCs by flow cytometry
Spheres were collected by centrifugation at 1500rpm for 5
minutes and mechanically dissociated to obtain a single-cell
suspension in 1ml 0.25% Trypsin/EDTA solution. Cells were
incubated at 37oC for 2 minutes and harvested by centrifugation
(2500rpm, 5 minutes). Pellet was rinsed in PBS solution, then
100µl FACS Fluid and 5µl CD133 antibody were added with the
ratio 1:200, respectively. This solution was incubated on an
orbital shaker at room temperature for 15 minutes or at 4 oC for 30
minutes. Centrifugation (2500rpm, 5 minutes) was performed to
harvest single cells, then cells were resuspended in 500µl FACS
Fluid solution. Cell sorting was performed on FACS Calibur flow
cytometer (BD Science).
2.2.2.2.3 Determination of Nestin expression of candidate
NSCs by immunocytochemistry
Spheres were preincubated in 1ml 1% BSA solution at
room temperature for 10 minutes, followed by washing with PBS
and centrifugation (1500rpm, 5 minutes). These neurospheres
7
were fixed by FCM fixation buffer and incubated on a rotator at
room temperature for 30 minutes, then they were washed and
centrifuged (1500rpm, 5 minutes) before being permeabilized in
1ml FCM permeabilization buffer for 5 minutes. Buffer was
removed by centrifugation (1500rpm, 5 minutes) and spheres
were resuspended in a solution containing anti-Nestin antibody
labeled with FITC and Hoechst 3342 at room temperature for 60
minutes or at 4oC overnight in dark conditions. Spheres were then
harvested by centrifugation, resuspended in 1ml PBS and plated
in 24-well plate for fluorescently microscopic observation.
2.2.2.3 Differentiation to astrocytes and GFAP expression
* Differentiation:
Spheres were harvested by centrifugation at 1500rpm for 5
minutes and resuspended in 2ml differentiation medium.
Suspension of cells was cultured at 37oC, 5% CO2 for 10 days.
* Immunocytochemistry:
Differentiation medium was removed and cells were fixed
by 100µl FCM fixation buffer, followed by incubation on a
rotator at room temperature for 30 minutes. After fixation, cells
were stained by anti-GFAP antibody labeled with Rhodamine and
Hoechst 33342 at room temperature for 60 minutes or at 4oC
overnight in dark conditions. Fluorescent microscopy was
employed to observe stained cells.
8
2.2.3 Activity 3: Evaluation effects of Ganoderma lucidum on
cell proliferation
Neurospheres, of which diameters were 100-250µm long,
were plated in 96-well plate (1-2 spheres/well) and cultured in
NSC medium modified with G.lucidum extract at different
concentration: 0µg/ml (Control), 100µg/ml, 500µg/ml and
1000µg/ml (37oC, 5% CO2). Each treatment was repeated 3 times.
The experiment with 1 factor was arranged randomly.
Criteria of evaluation: changes in neurosphere sizes were
recorded every 24 hours for 4 days by observing under inverted
optical microscope.
2.2.4 Data analysis
Changes in neurosphere sizes were reported as mean ±
standard deviation (SD) with 95% of confidence. Differences
among means were analyzed by T-test, using Microsoft Excel
2007 software.
9
CHAPTER 3: RESULTS AND DISCUSSION
3.1 Isolation and culture of candidate NSCs from embryonic
mouse brain
3.1.1 Isolation and primary culture of candidate NSCs
Among 14 samples, 10 samples were successfully cultured
with the proportion of 72%. NSCs isolated from the 13.5-15.5 day
post coitum embryonic brains, especially E14 cells, show the best
rate of proliferation and sphere formation since during this period,
receptors for growth factors dominantly expressed, resulting in
rapid cell growth. Tropepe et al. (1999) also demonstrated that
more spheres formed when E14.5 and E15.5 NSCs were cultured
in medium modified with EGF and FGF. Moreover, since cells
derived from E18 brain committed to differentiate to some extent,
which led to the decrease in receptor numbers as well as
proliferation capacity, less sphere were observed from these
samples. Although E8.5 cells showed rapid proliferation and more
spheres formed, contaminated fibroblast, which derived from
mouse scalp, could excrete factors signaling cell adhesion and
intense differentiation.
In culture samples, many types of cell were observed,
including, erythrocytes, fibroblasts, NSCs, some other cell types
and lipid. However, those non-neural cells were eliminated after
passages.
Isolated
candidate
NSCs
showed
round-shape
morphology, bright
nucleus and sized between 8-12µm
(Schumacher, 2003).
Such cells grew in NSCs medium and
formed cluster within 24 hours of culture. After 3-4 days, lipid
and some other dead cells which could not grow in NSCs medium
10
aggregated into opaque clumps, in which several clusters of
candidate cells (approximate 50µm) stuck. 100-150µm spheres
were observed after 5-7 and reached the size of 250µm in day 10
of culture since cells in such sphere grew and proliferated,
resulting in more daughter cells. Similar results also presented by
Ge et al. (2012).
(a)
(c)
(e)
Figure 4.2’. Sphere formation from primary culture.
(a) Isolated cells showed round-shape, red arrows: NSCs, blue
arrows: erythrocytes (04/08/2013). (c) Opaque clumps after 4
days (04/04/2013). (e) Sphere after 11 days (04/19/2013).
3.1.2 Secondary culture
After 2-3 days, cells were passed to other wells. Passage
helped remove toxic metabolites, dead cells, provide nutrients and
space for cell growth. When being mechanically dissociated,
opaque clumps were removed and clusters of 3-4 cells were
observed. Such clusters formed more small spheres within 24
hours and achieved clear round-shape after 3-4 days. Those
spheres then continuously increased in size and had round,
compact structure after passage 3 or 4. At the same time, other
non-neural cells, which could not grow in NSC medium, died and
11
were removed, proving that during passages, only neural-like
cells could survive and become dominant. After passage 7 or 8,
cells in spheres died, leading to the lysis of protein linkages and
the loss of spherical structure. The death of cells was shown to be
related to caspase-3 (Milosevic et al., 2004), which is responsible
for tumor suppression.
3.2 Determination of characteristics of candidate NSCs
3.2.1 Neurosphere assay
Spheres of passage 4 were mechanically dissociated and
cultured in 24-well plate. After 24 hours, small clusters formed
and 50µm spheres were obtained in the next 2-3 days. Spheres
became more compact and had the diameters ranging from 100200µm after 5-7 days and 250-400µm after passage 2-3.
According to Reynolds and Weiss (1992), when single cells
derived from SVZ were cultured in serum-free medium in the
presence of EGF, they divided and initially adhered to the plate
and detached to form spheres of proliferating cells after a few
days. To assess self-renewal of cells, spheres were mechanically
dissociated and cultured again in medium containing EGF to
determine if single cells could form new spheres. Bez et al.
(2003) indicated that 80% of cells derived from primary
neurospheres could form secondary and tertiary neurospheres.
However, low density of cultured cells leads to less forming
spheres in comparison to high cell density since cell-cell
interaction could induce excretion of growth factors (Pastrana et
al., 2011).
12
3.2.2 Expression of markers of candidate NSCs
3.2.2.1 Sox1 expression of candidate NSCs
Sox is a transcription factor in developing brains and helps
maintain undifferentiated manner of NSCs by inhibition of
neuronal differentiation. According Venere et al. (2012), Sox1
was an efficient marker for detecting NSCs derived from animal
SGZ.
Figure 4.6. Electrophoresis of RT-PCR products.
1: ladder, 2: GAPDH, 3: Sox1 (04/27/2013)
As stated in Appendix 1, Sox1 gene sized approximately
165bp, which was similar to 170bp band observed in well 3,
revealing the expression of Sox1 in these candidate cells.
According to Bhaskar et al. (2005), Sox1 was expressed in most
of E14 NSCs, and Chen et al. (2009) demonstrated that such
amplified products of such gene in several animals ranged around
200bp.
13
The 300bp band on agarose gel was the housekeeping gene
GAPDH, which was responsible for basic function of cells and
popularly used in comparison of expression levels of different
genes.
3.2.2.2 CD133 expression of candidate NSCs
Firgure 4.7. 2D- dot plot
Figure 4.8. Histogram
(04/28/2013)
(04/28/2013)
Flow cytometry histogram revealed that 59.51% of
candidate cell was CD133 positive. Integrated data of all above
mentioned results indicated that such cells were NSCs (Figure
4.8). However, since cells were harvested after second passage,
undesired cells were not completely eliminated, resulting in low
proportion of CD133+ cells. In addition to NSCs, neurospheres
consisted of glial cells and neural progenitors at different stages
of mitosis and differentiation which affected flow cytometry
results.
14
3.2.2.3 Nestin expression of candidate NSCs
Stained spheres showed green fluorescence of FITC and
blue fluorescence of Hoechst 33342, indicating the presence of
cells with nuclei in neurosphere structure, and such cells were
Nestin positive (Figure 4.9). In contrast, less cells located in outer
layer expressed this protein.
Nestin is a type of intermediate filaments in neural stem
cell cytoskeleton; however, gene encoding for this protein is
down regulated during cell differentiation, thus such protein was
less expressed in larger sphere, which correlated with more
differentiated cells. Due to compact structure of neurosphere,
which might limit the access of labeled antibody, fluorescent
emission of inner layers was not clearly observed.
Figure 4.9. Nestin expression of NSCs (05/03/2013)
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