Article

Induction of Mesenchymal stem cells into Neuronal Cells via two formulas

Abstract and Figures

Mesenchymal stem cells (MSCs) are appropriate source of multipotent stem cells that are valuble source for cell-based therapies. They can be differentiated into neuron cells under appropriate conditions. We aimed to conduct a comparative study to evaluate the best differentiation method of MSCs induction into neural cells. We tested two types of neural differentiation formulas. The first formula was butylated hydroxyanisole (BHA), and the second formula was β-mercaptoethanol (BME). This study was done by using three different neural markers: nestin (NES) as immaturation stage marker, neurofilament light-chain (NF-L) as an early neural marker, and microtubule association protein (MAP-2) as maturation marker. These markers represented the different neurogenesis stages starting from mesenchymal stem cells (as undifferentiated cells), neural stem cells production stages, and neuron cells (as differentiated cells). The results of immunocytochemistry analysis of both BME and BHA methods showed that both methods were successful in produced neuron cells in culture with a different efficiency. However, the best formula used was BME which induces fast neural differentiation. There was upregulation to NES protein significantly within first hour of exposure compared with BHA method which needs longer time. NF-L upregulated significantly later was compared to undifferentiated MSCs. The neural maturation marker MAP-2 expression increased in BME method with 29h while it needs 144h for BHA method. The results suggest using BME method as preferable neurogenesis induction method in regenerative medicine applications.
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Research Journal of Biotechnology Vol. 14 (Special Issue I) March (2019)
Res. J. Biotech
265
Induction of mesenchymal stem cells into neuronal cells
via two formulas
Mohammad Maeda H.1*, Al-Shammari Ahmed M.1, Almzaien Aous K.1, Hamad Mohammed A.2, Hassan Ayman A.1,
Shaker Hiba K.1 and Yaseen Nahi Y.1
1. Experimental Therapy Department, Iraqi Center of Cancer and Medical Genetic Research, Al-Mustansiriyah University, Baghdad, IRAQ
2. Department of Biotechnology, College of Applied Science, University of Fallujah, Anbar, IRAQ
*maeda.mohammad@iccmgr.org
Abstract
Mesenchymal stem cells (MSCs) are appropriate
source of multipotent stem cells that are valuble source
for cell-based therapies. They can be differentiated into
neuron cells under appropriate conditions. We aimed
to conduct a comparative study to evaluate the best
differentiation method of MSCs induction into neural
cells. We tested two types of neural differentiation
formulas. The first formula was butylated
hydroxyanisole (BHA), and the second formula was β-
mercaptoethanol (BME). This study was done by using
three different neural markers: nestin (NES) as
immaturation stage marker, neurofilament light- chain
(NF-L) as an early neural marker, and microtubule
association protein (MAP-2) as maturation marker.
These markers represented the different neurogenesis
stages starting from mesenchymal stem cells (as
undifferentiated cells), neural stem cells production
stages, and neuron cells (as differentiated cells).
The results of immunocytochemistry analysis of both
BME and BHA methods showed that both methods
were successful in produced neuron cells in culture
with a different efficiency. However, the best formula
used was BME which induces fast neural
differentiation. There was upregulation to NES protein
significantly within first hour of exposure compared
with BHA
method which needs longer time. NF-L
upregulated significantly later was compared to
undifferentiated MSCs. The neural maturation marker
MAP-2 expression increased in BME method with 29h
while it needs 144h for BHA method. The results
suggest using BME method as preferable neurogenesis
induction method in regenerative medicine
applications.
Keywords: Comparative study, stem cells, neuron cells,
NES, NF-L, MAP-2.
Introduction
Stem cell technologies have attracted many investigators in
the past two decades. The progress in embryology,
hematology, neurology, skeletal biology and many other
disciplines in stem cells research has led to a complete
understanding of these cells. It is centered on the isolation,
characterization, differentiation of in vitro and in vivo
studies and therefore uses it in the treatment of a variety of
diseases24,30,47. The stem cells therapies are potential clinical
treatment for neural regenerative diseases. Although these
therapies are attractive option to reverse neural tissue
damage and to recover neurological deficits, it is a limited
treatment because the conditions of the nervous system
cannot be completely recovered after treatment12,46.
Also, these therapies are still under development so as not to
show significant treatment effects in clinical settings40.
Therefore, for improving the treatment of neurodegenerative
therapy, differentiated cells from mesenchymal stem cells
(MSCs) sources have been investigated throughout the past
two decades for use as an alternative source than
undifferentiated stem cells 5. Many research demonstrates
that the MSCs (as one of the primary sources of stem cells)
can be differentiated into different neural cells, and then used
in clinical treatments4,27,33,35,45,46,48,50.
These differentiated cells can be used in treatments and
generate cells for transplantation therapies in number of
central nervous system (CNS) injuries and
neurodegenerative diseases (as cell-based therapies) in Iraq
and many others countries, which can replace these lost cells
or repair their damaged areas, therefore provide functional
recovery15,20. Until now, many protocols induced neural
differentiation of MSCs in vitro 37. It was about isolation,
culture conditions, and molecular characterization, but in
general the results of these studies lack consistency and
comparability. Defining the best formulas used for neural
induction and also defining the exact time of neural
expression during the differentiation of MSCs towards
mature neuron which will be useful to use in neural disease.
For all the above reasons the primary objective of this study
is to select the best differentiation formula that induces
neural cells from bone marrow MSCs.
Material and Methods
Induction of mesenchymal stem cells in culture: Bone
marrow cells were prepared and maintained as described by
Freshney13. In the animal house, donor male mice were
killed by cervical dislocation (3- 6 weeks old, the protocol
agreed by the ICCMGR animal care and use committee).
The fur sterilized using 70% alcohol and the femurs and
tibias were released and transported into a Petri dish
containing serum free media MEM (Minimum Essential
medium) (US Biological, Massachusetts USA)
supplemented with 500µg/ml streptomycin and ampicillin
(Capricorn-scientific, Germany)1. In the laboratory, under
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sterilized conditions, the femurs and tibias were clean off
from remaining muscle tissues (with sterile surgical tools)
and washed for three times using Phosphate Buffer Saline
(PBS), then hold the femur with forceps and cut both ends to
flush out the marrow cells.
The marrow was dispersed to a suspension by pipetting.
Lastly, the cell suspension was dispensed into 25-cm2 tissue
culture flasks (Thermofisher scientific, USA) and incubated
at 37˚c in humidified 95% air and 5% CO2. The cells were
allowed to adhere, and non-adherent cells were washed out
(with serum-free medium) after 24h. The nonadherent cells
were removed by changing culture medium each 2-3 day
until the cultures were getting hold of developing colonies
of adherent cells (about 5-7 days) to formed monolayer's
cells. After that, cells were recovered after getting
monolayer cells using 0.25% trypsin-EDTA (US Biological,
Massachusetts USA). The passage one (P1) cells began to
proliferate and formed a monolayer after 3-5 days 19.
Protein detection of MSCs in culture: The MSCs were
seeded in 8-well tissue culture chamber slide (IWKA,
Japan). The cells were allowed to developing a monolayer
for phenotyping using immunocytochemistry assay. The
cells were fixed with 4% paraformaldehyde in PBS for 10
minutes, washed with PBS and left to dry; the cells were
stained with five specific markers immunophenotypic
analysis. The markers tested were: CD90 (1:100; Mouse
anti-human antibody, US biological, USA, C2441-06),
CD105 (1:100; Mouse anti-human antibody, US biological,
USA, C2446-50B), CD44 (1:100; Rat anti-mouse antibody,
US biological, USA, C2398-01T), CD34 (1:100; Goat anti-
human antibody, Santa Cruz Biotechnology, SC-7045), and
Nestin (1:50; Mouse anti-rat antibody, Santa Cruz
Biotechnology, Europe, SC-58813). Mesenchymal stem
cells were blocked in a humidified chamber with 1%
hydrogen peroxide (H2O2) for 10 minutes and then incubated
with 1.5% blocking serum for 30 minutes at room
temperature (the kit from ImmunoCruz mouse ABC Staining
System, SC-2017, Santa Cruz Biotechnology, Europe).
The primary antibodies were incubated overnight at 4ºc in a
humidified chamber. After extensively washed with PBS,
the biotinylated secondary antibody (anti-mouse for all CD
markers except in CD34 anti-goat) was incubated for 30
minutes, washed 2-3 times with PBS. The avidin was
conjugated to horseradish peroxidase (HRP) and added for
each slide and incubated in a humidified chamber for another
30 minutes, then washed 2-3 times with PBS. For visualizing
the peroxide, liquid DAB chromogen solution mix was
added for each slide for 20 minutes at room temperature,
washed extensively with PBS and counterstained in
Hematoxylin stain for 30-60 second. The slides were
mounted with DPX and then were inspected by using a light
microscope and photographed by using a digital camera 2.
Viable Cell Counting: The cells were counted to study the
differentiation efficiency before and after induction. Viable
cell counting on studied cells was accomplished using
Trypan blue exclusion. Dead cells, unlike viable cells, took
up the dye within a second making them easily
distinguishable from viable cells under the microscope. The
protocol was done according to Janke et al22. One part of the
cell suspension (0.2 ml) was added to one part on trypan blue
stain (0.2ml) and add eight parts (1.6 ml) PBS to complete
the final volume (2 ml). Then 20 μl of sample was
transferred to the edge of the cover - slip, running into the
counting chamber. Finally, the cells were counted in the four
1mm center squares which included a separated count of
viable and non-viable cells, this counting was done by using
a light microscope under 40X objective.
The observation was done by using a light microscope under
40X objective. The cell concentration (cell/ml), cell viability
(%), and total cell count were calculated from the following
equation11,14:
C= n x d x l0000
where C= Cell concentration (cell/ml), n= number of
counted cells, d= dilution factor diluted by with free serum
media = l0). Total Cell Count = C (cell/ml) x the original
volume of fluid from which the cell sample was removed.
Neural induction in culture: Subconfluent cultures of P1
MSCs were used to induced neural differentiation by using
two different differentiation formulas as follows:
Butylated hydroxyanisole (BHA): Santa Cruz
Biotechnology, USA was modified54 for 24, 25, 27, 29, 48,
96, and 144h. These exposure times were divided as
following: pretreated in MEM media with 20% FBS and ten
ng/ml bFGF (USbiological, USA) as preinduction media for
24h exposure. Then another exposure was used (of these
cells as another formula) to induce neuronal differentiation
(as post induction media) by using MEM media without FBS
(serum free media), 2% DMSO (Santa Cruz Biotechnology
(USA)), and 200 μm BHA (Santa Cruz Biotechnology,
USA) for 1, 3, 5, 24, 72, and 120 hrs. as modified from
Kalcheva et al23.
β mercaptoethanol (BME): Santa Cruz Biotechnology,
Europe, USA for 1, 6, 12, 18, 24, 25, 27, 29 and 34 hrs.
exposure times was used; these exposure times were divided
as follows: pretreated in MEM media with 20% FBS and 1
mM BME as preinduction media for different exposure
times (1, 6, 12, 18, and reaching to 24 hrs. exposure). Then
another exposure was used (of these cells as another
formula) to induce neuronal differentiation (as post
induction media) for all these exposure times by using MEM
media without FBS (serum free media), and 5 nM BME for
different exposure time (1, 3, 5, and 10 hrs.) as modified
from Li32.
Morphological Study by H and E Staining: The
undifferentiating (MSCs) and differentiating cells (neural
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cells) for each differentiation methods BME (for 24, 25, 27,
29 and 34 h exposure time) and BHA (for 25, 27, 29, 48, 96
and 144 h exposure time) were used for morphological study
using Hematoxylin as nuclear stain (SYRBIO, Syria) and
Eosin as cytoplasmic stain (SYRBIO, Syria) as modified by
Lim et al33 as the follows: The MSCs (P1) were cultured in
multi-well tissue culture plates (8-wells), then the
differentiation was induced in all different exposure time of
each BHA and BME differentiation methods compared with
MSCs (as control), the slides were washed with PBS after
exposure.
After that, the slides were stained with Hematoxylin for 5
minutes, and washed with distilled water for 2 minutes. After
that the slides were stained with Eosin for 2.5 minutes,
dehydrated with ethanol 95% for 1 minute and ethanol
absolute (100%) for 4 minutes. Finally, cleared with xylene
for 2 minutes, washed with ethanol absolute for 2 minutes,
and mounted with DPX. All slides were photographed by
light microscope camera.
Protein detection of differentiated cells in culture: The
cells were stained for NES (1:50; Mouse anti-rat, Santa Cruz
Biotechnology, Europe, SC-58813), and NF-L (1: 100;
Mouse anti- porcine, US biological, USA, 031403) as
immature neuronal cells markers, and for MAP-2 (1:50;
Mouse anti-human, Santa Cruz Biotechnology, SC-74421)
as mature neuronal marker. Then stained using staining kit
(ImmunoCruz mouse ABC Staining System, Santa Cruz
Biotechnology, Europe, SC-2017).
Statistical analysis: All data were statistically analyzed
using One Way ANOVA and LSD test in IBM SPSS
Statistics Software (version 20) and the difference of means
was considered significant at p<0.05 for ICC assays (as
average percentage means).
Results and Discussion
Induction of Mesenchymal stem cells in culture:
Mesenchymal stem cells were cultured after isolation
method in tissue culture flasks in MEM medium
supplemented with 20% FBS (as culturing and maintenance
media) and left for 24 h., then maintained for use in this
study. After 24 h. culture, the results showed that only a few
cells attached to the plastic culture flasks sparsely and
formed adherent cells (which represent MSCs) with the
rounded or spherical shape. While the non-adherent cells
(which represent erythrocyte cells and another cell type)
were discarded by the first medium usually changing after
24 or 48 h. (Fig. 1A). After that, the adherent cells began to
proliferate. Moreover, 2-3 days after cultivation, the
numerous fibroblasts like-cells could be observed and
gradually grow to form small individual colonies displaying
as fibroblast-like cells morphology.
In addition to, small round cells can also be seen (Fig.1B).
Mesenchymal stem cells are categorized by their capacity to
form colonies including spindle-shaped cells deriving from
a single cell. The number of cellular colonies with different
size has increased. After few days, the cells in large colonies
appeared more densely distributed and showed a spindle-like
shape, as the growth of cells continued, colonies gradually
expanded in size with adjacent ones and interconnected with
each other.
Figure 1: Culture and maintenance of MSCs (A): after 24 h. (B): after 72 h. note that some of cells adherent and
began to elongate. (C): after 5-6 days of culturing (monolayer cells as P0), note that the colonies interconnected with
each other and reaching a confluent stage. (D): after the first passage (P1), the culture of MSCs as revealed under an
inverted microscope for 10 X for each of culture respectively.
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Then when the primary cells as passage zero (P0) grow to
70-80% confluence after 5-6 days, they were ready to
passage for the first time by sub culturing (Figure 1C). The
subculture done was poured out in the media, treated with
0.25% trypsin-EDTA and cell suspension passed into new
tissue culture flasks under the same conditions at a split ratio
1:2 for the first passage culture. The MSCs began to grow
and formed colonies then expanded, and after few days from
P1, homogeneous layer of fibroblastoid-like cells occupied
the whole plastic surface (Fig.1D). The cells can be reseeded
in same conditions for the second passage culture, and these
adherent cells could be readily expanded in vitro by
successive cycles of trypsinization, seeding, and culture
every 5-7 days without visible morphologic alteration.
Viable Cell Counting: To study the differentiation
efficiency before and after differentiation (for the different
exposure times used in this study), the cell counting and
viability were determined in this study. The results showed
that the viability of the cells in each primary culture P0, P1,
or even P2 was suitable to enter the next experiment with
high percentages of viability cells (ranging between 75-
90%) and with high cells counting (table 1). Therefore, the
MSC number in this study seemed to be enough for in vitro
study.
Phenotyping detection of MSCs in culture: With the aim
of study of the nature of the fibroblast-like cells (MSCs) in
culture, the P1 of MSCs was re-cultured in 8-well tissue
culture chamber slide and left to grow and expand within 2-
3 days to form a homogeneous layer occupying the whole
slide surface. Then the culture medium aspirated and the
cells were fixed to prepare for labeling against MSCs-related
surface antigens which are as follows with their cellular
distribution (immunocytochemistry technique) for five
markers: CD105, CD90, CD44 as positive markers and
CD34 and NES as negative markers.
The results showed that the immunophenotypic
characterization of the cultured MSCs stained with CD34
and NES showed negative results through stained whole
cells in culture with blue color (from hematoxylin stain) for
each cell surface and nucleus, and the identification
percentages were 5% and 10.5% for each CD34 and NES
respectively (Figure 2 AD). These results indicated that
these cells were not from the hematopoietic origin (which
represent CD34) or differentiated cells (which represent
NES), but they were MSCs (Table 2). Slides that stained
positive for CD44, CD90, and CD105 showed brown
cytoplasm and with blue nucleus. CD44 expressed in 89% of
cells (Figure 3A, B) (Table 2).
Cells stained with CD90 and CD105 antibodies showed dark
brown color for each cytoplasm and nucleus in 94% of cells
(Figure 3 CF) compared with negative control which takes
the blue color only (Figure 2 AD) and table 3. Therefore,
these results indicate that they retain the phenotype of MSCs.
The results showed that MSCs were negative for CD34 and
NES which take the blue color (from Hematoxylin stain),
which indicates that these cells are not of hematopoietic
origin, but they were MSCs.
Table 1
Cells counting in the experimental study.
Cells type
tissue culture flask
3
Count in 25 cm
MSCs (as a primary culture)
6
8.33 x 10
MSCs (P1)
6
2.50 x 10
MSCs (P2)
6
2.54 x 10
Differentiated cells using BME
24 h
6
1.90 x 10
25 h
6
1.40 x 10
27 h
6
1.45 x 10
29 h
6
0.90 x 10
34 h
6
0.73 x 10
Differentiated cells using BHA
24 h
6
2.05 x 10
25 h
6
10 x1.20
27 h
6
1.00 x 10
29 h
6
1.00 x 10
48 h
6
0.75 x 10
96 h
6
0.60 x 10
144 h
6
0.35 x 10
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Figure 2: Immunophenotypic analysis of MSCs at the first passage revealed under the light microscope show that the
MSCs were negative by cell stained with blue color. (A and B): CD 34. (C and D): Nestin. Note that the two markers
showed with 10 and 40X respectively.
Table 2
ICC results for different surface markers of cultured MSCs.
Identification percentages (%)
Positive markers
89.0
95.0
95.0
Negative markers
5.0
10.5
Figure 3: Immunophenotypic analysis of MSCs at the first passage revealed under light microscope show that the
MSCs were positive cells which stained with brown color. (A and B): CD44, (C and D): CD90, (E and F) CD105. Note
that all CDs showed with 10 and 40X respectively
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Neural induction in culture: The differentiation process of
stem cells occurred through a tremendous genetic nest by
changing their expression levels by up and down of protein
regulation starting from stem cells toward neuron cells
formation 2. The results showed that MSCs from mouse bone
marrow were successfully induced neural differentiation by
using the two different formulas used in this study. As the
first differentiation method by BHA, sub confluent cultures
of P1 MSCs were pretreated for 24 h in MEM medium with
20% FBS and ten ng/ml bFGF (as preinduction medium).
Then neural differentiation (as post induction medium) was
induced with MEM medium without FBS (serum-free
medium), 2% DMSO, and 200 μm BHA.
The results showed that the MSCs in the induction medium
(for 24 h exposure) showed to be more elongated and
increased in the proliferation rate (Figure 4 A) compared
with undifferentiated MSCs (as negative control); these
results proved the proliferation of the stem cells in culture
with the present of b-FGF (as proliferation factors) in the
preinduction medium. Then, after exposure to BHA with
DMSO for different time intervals (as postinduction
differentiation medium), the MSCs started to be more
elongated and increased in size (including the nucleus and
cytoplasmic content) (figure 4 B, C). Then starting from 29
h, the differentiated cells formed cells branched, and then
increased the cells branched for each 48 and 96 h exposure.
Towards 144 h showed the formation of the cell body,
dendrites, and axon as the neuron cells morphology (figure
4 D-G).
As the second differentiation formula, the sub confluent
cultures of P1 MSCs were using to induced neural cells
differentiation by using BME for different exposure time (1,
6, 12, 18, 24, 25, 27, 29 and 34 h). This method was divided
into two-step, pre-treated step in MEM medium with 20%
FBS and 1 mM BME for different exposure times (1, 6, 12,
18, and 24 h), then transport these cells in to post-treated step
by using MEM medium without FBS and 5 mM BME for
different exposure time (1, 3, 5, and 10 h).
Figure 4: MSCs after induced differentiation by BHA, the pointer arrows were showed the elongation of cells and
branched form towards neural cells as revealed under an inverted microscope, all figures showed in 10X, the figures
(A-G) presented (24-144 h) exposure time to differentiation medium.
D
E
F
G
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The results of preinduction medium (1, 6, 12, 18, and 24 h
exposure time) indicated that the differentiated cells were
shown to be more spherical than MSCs, these changes were
started from 24 h (Figure 5 A) reaching to the post-induction
medium (25, 27, 29, and 34 h). These changes occurred by
increase in cells size, forming cells branches, then formed
cell body and dendrites towards neuron cells morphology
(Figure 5 B-E). The BME as antioxidants reagents is known
to influence metabolism and promote cell survival in some
cell culture systems. Nevertheless, their effects are on the
modulation of bone cell differentiation in vitro 18. Also, it
was a successful differentiation method as neural inducer
within a few hours, throw their expression of many neural
markers through differentiation process11,19,27,35,43,44,54. Other
studies used BME with other neural inducers as pri-
induction reagents such as with retinoic acid 38, with retinoic
acid and creatine 9, with b-FGF 54, or with different
supplemental reagents 28. Therefore, the BME was
successful in induced neuron cells in this study compared
with other formula used in this study.
Morphological Study by H and E Staining: Mesenchymal
stem cells and induced neuron cells morphology were
studied for BHA differentiation method at specified intervals
(24, 25, 27, 29, 48, 96, and 144 h exposure time) by staining
with Hematoxylin (as a nuclear stain) and Eosin (as a
cytoplasmic stain). The results showed that the MSCs after
differentiation started to be more spherical in cell shape with
the dark red color of the cells (from Eosin stain) and the
nucleus seems to be bigger with dark blue color (from
Hematoxylin stain) through the differentiation process
(figure 6). Then many cells formed branched as neural cells,
and these branched increased in length and formed a nest of
cells branched connecting with each other through the
different exposure times (figure 6 C-P) compared with the
classical shape of MSCs (figure 6 A and B). The
undifferentiating (MSCs) and differentiating cells (neural
cells by using BME for 24, 25, 27, 29, and 34 h exposure
time) were used to study the morphological changing
through using Hematoxylin (as a nuclear stain) and Eosin (as
a cytoplasmic stain).
The results showed that the MSCs after differentiation
started to be more spherical with the dark red color of the
cells (from Eosin stain) and the nucleus seems to be bigger
with dark blue color (from Hematoxylin stain) through the
differentiation process (figure 7). Then many cells formed a
branched as neural cells, and these branched increased in
length and formed a nest of cells branched that connect with
each other through the different exposure time (figure 7 C-
L) compared with the classic shape of MSCs (figure 7 A and
B).
Figure 5: MSCs after induced differentiation by BME, the pointer arrows were showed the spherical shape of cells
and branched form towards neural cells as revealed under an inverted microscope; all figures showed in 10X. The
figures (A-E) presented (24-34h) exposure time to differentiation medium.
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Figure 6: H and E study, the results were showed the differentiation of MSCs by BHA towards neural cells through
different exposure times as revealed under light microscope, all figures showed in 10X and 40X respectively. The
figures (A-P) presented (MSCs- 144 h) exposure time to differentiation medium.
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Figure 7: H and E study, the results showed differentiation of MSCs by BME towards neural cells through different
exposure time as revealed under light microscope, all figures showed in 10X and 40X respectively. The figures (A-L)
presented MSCs-34 h exposure time to differentiation media.
Protein detection of differentiated cells in culture: The
MSCs were processed for immunocytochemistry assay
which represents undifferentiating MSCs (as negative
group) and the neural cells after using BHA for 24, 25, 27,
29, 48, 96 and 144 h exposure time by using three
differentiation markers NES as NSCs and immaturation
marker, NF-L as an early neural marker, and MAP-2 as
maturation neuron marker as showed in figures 8, 9,10 and
11. The ICC results showed that the NES protein founded in
the MSCs in deficient levels (10.5%), at the beginning. NES
expression was increased significantly (p<0.05) through
neural differentiation intervals. It started from low (12.3%)
at 24h exposure time (as preinduction medium) and
continued to increase significantly (p<0.05) through
different exposure time (25, 27, 29, and 48 h) as
postinduction differentiation medium with 45.1, 50.1, 49,
and 61.2% respectively. The highest proteins level was at
48h of exposure. Then, NES protein expression was
decreased through the next 96 and 144h (with 39.9 and 43.8
% respectively) compared with the undifferentiating MSCs
as a negative control as shown in figures 8 and 11.
Nestin is an intermediate filament protein (IFPs) used in this
study for evaluation of the two differentiation formulas was
expressed in dividing cells during the early stages of
development in the CNS, peripheral nervous system, adult
brain and in myogenic and other tissues3,28. Also, it is
expressed by many types of cells during development,
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although its expression was usually transient and does not
persist into adulthood during differentiation. This gene was
down-regulated and replaced by tissue-specific intermediate
filament proteins such as the NES protein of NSCs in the
adult mammalian forebrain replaced by neurofilament and
glial fibrillary acidic protein (GFAP) in each neuron and
glial cells39. Also during neuro- and gliogenesis processes it
was replaced by cell type-specific intermediate filaments
neurofilaments NFs and glial fibrillary acidic protein
(GFAP) 36.
In the generation of neuron cells from neurosphere culture,
nestin expression was down regulated after differentiation
compared with neurospheres cells which were expressed in
high levels 55. This research proved the expression of NES
protein during the neurogenesis process which matched with
this study. Correspondingly, the results showed that NF-L
protein levels was shown to be increased significantly
(p<0.05) started from 25 h exposure time (27.5%) reaching
to different exposure time 27, 29, 48, 96, and 144 h (with
31.4, 39.6, 69.8, 73.1, and 86.7% respectively), with higher
protein levels at 144 h taking dark brown color compared
with MSCs (as negative control) which recorded low protein
levels (15.1%) as shown in figure 9 and 11.
Neurofilament protein was one of the essential cytoskeletal
proteins of neurons, and it was a biomarker for
neurodegeneration 14, the subtype light-chain of the
neurofilament group (NF-L) represented the most abundant
and also most soluble neurofilaments subunit compared with
other subunits 44.
The NF-L subunit was found in significant amount in blood
and serum compared with other two type (M and H-chain)
and also used as a biofluid source which was easily
accessible for longitudinal studies of neurodegenerative
neurological diseases 14. Also, the expression of each class
of neurofilaments was changing during different
developmental stages. For instance, NF-M and NF-L
subunits were expressed early during embryonic
neurogenesis while NF-H appears later during the postnatal
period in rats7,29. Therefore, it was indicated that NF-L was
expressed in the earlier stage of neurogenesis and it is a good
choice for use in the study of the neurogenesis stages during
in vitro differentiation.
The results of this study showed very low levels of MAP-2
protein through the differentiation process compared with
both NES and NF-L used in this study, which started to
increase with significant difference level (p< 0.05) starting
from 25, 27, 29, 48, 96 and 144 h exposure time (with 13.9,
9, 6.2, 16.4, 21.8 and 23.9% , respectively) with higher
protein levels in 144 h compared with no protein levels in
MSCs (as negative control) with 0.5% which takes the blue
color as shown in figure 10 and 11.
Figure 8: ICC detection results of MSCs after induced differentiation by BHA, the results showed increased of NES
protein through different exposure times as revealed under a light microscope, the figures (A-H) represented (MSCs-
144 h) exposure time.
B
C
D
F
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Also, the analysis of protein expression of MAP-2 has been
used for neural confirmation 23,25. These mature specific
markers played a role in the stability of axons, and neuronal
cell bodies throw differentiation process 22. Therefore
maturation markers were absent or with low expression in
the early stage of neural differentiation in our study. In
general, the MAP-2 gene expression is weak in neural
precursors, but it increases during neuron development
process. Also, its expression is confined to neurons and
reactive astrocytes 6,49.
Therefore, MAP-2 was expressed in the early and immature
neural stage but at very low levels, therefore it matched with
the results of this study. In general, the ICC results for BHA
showed increase in the protein expression levels of each
NES and NF-L protein but in low levels started significantly
from 25 h and increased through the 27, 29, 48, h with the
higher proteins expressions in 48 and 144 h for each NES
and NF-L respectively compared with MAP-2 protein which
showed less protein expression levels. The ICC results of
using BME as the second differentiation formula showed
that the NES protein was found in the MSCs (ac control
group) in very low levels (10.5%) (in positive results, the
cytoplasm of the cell took brown color from the DAP stain).
Then its level increased significantly at p<0.05 through
neural differentiation starting from 24 h exposure time with
61% which displayed the brown color. After that, NES was
upregulated significantly (p<0.05) through 25 and 27 h
exposure time (with 68.5 and 86.3%) with morphological
changing of cells towards neural cells, and with the highest
protein levels in 27 h, then NES protein was decreased its
levels in 29 h with 61.8% significantly (p<0.05) compared
with control with highly morphological changing towards
neural cells as showed in figure 12 and 15.
The results of this study showed that the NF-L protein
expression increased significantly (p<0.05) through the
differentiation process (24, 25, 27, and 29 h) exposure time
which recorded 32.2, 65.9, and 80.8% through increase of
the dark brown color in the differentiated cells reaching to
the highest protein levels in 27 h exposure time which take
the dark brown color.
Figure 9: ICC detection results of MSCs after induced differentiation by BHA, the results showed increase of NF-L
protein through different exposure times as revealed under a light microscope, the figures (A-H) represented (MSCs-
144 h) exposure time.
F
E
D
C
B
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Figure 10: ICC detection results of MSCs after induced differentiation by BHA, the results showed increased of
MAP-2 protein through different exposure times as revealed under a light microscope, the figures (A-H) were
represented (MSCs-144h) exposure time.
Figure 11: The proteins expression levels of ICC assay for NES, NF-L, and MAP-2 antibodies
of neural cells after induced by BHA.
*refer to significant effect at p>0.05.
Then its level decreased significantly at 29 h exposure at
p<0.05 with 66.4% compared with low protein levels in
MSCs with 15.1% (as negative control) as shown in figures
13 and 15. The results of this study showed very low levels
of MAP-2 protein compared with the other two types (NES
and NF-L) through the differentiation process, no protein
expression with the pre-induction medium and less protein
expression levels in post-induction medium were observed.
E
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Then started to be expressed from 27 and 29 h (12 and
29.7%) with significant difference level at p<0.05 through
the dark brown color compared with no protein levels in
MSCs with 0.5% (as negative control) which take the blue
color as shown in figures 14 and 15.
In general, the ICC results for BME showed increase in the
protein expression levels for each NES and NF-L protein
started at 24 hrs. (61% for NES and 32.2% for NF-L). NES
was 86.3%, and NF-L was 80.8% compared with
undifferentiating MSCs (as a negative control) with 10.5%
for NES and 15.1% for NF-L. The MAP-2 proteins showed
no protein expression in the pre-induction media. Less
protein expression levels were noted in post-induction media
which increased starting from 27 and 29h with 12% and
29.7% of protein levels percentages respectively with a
significant difference at level p< 0.05 compared with
undifferentiating MSCs (as a negative control) with 0.5% (as
shown in figure 15).
Results indicated the activity of BME in the neurogenesis
stages of MSCs towards the neural differentiation cells (by
induced the immaturation stage of neural differentiation
using BME), throw increase of the nestin protein levels
combined with neurofilament levels compared with low
protein expression in the MAP-2 protein (as maturation
marker) in all exposure time.
The results of this study proved that both NES and NF-L
markers used in this study were good choice for evaluating
the differentiation formula. Because both markers were
involved in the neural induction and fully neuronal
differentiation, it threw increase in the protein expression
levels of each NES and NF-L protein (as an early neural
marker) compared with MAP-2 protein (as late neural
markers) which showed less protein expression levels for
each BHA and BME which indicated (according to the
results above) that these markers worked together
respectively and therefore it was a good choice to study the
neural differentiation.
Many studies matched with the results of this study through
increasing of many neural markers such as NES, NF-L, and
in the late stage increasing of MAP-2 after induced neural
differentiation by using BHA differentiation formula11,27,49.
Also, other studies indicated that differentiated and
undifferentiated of MSCs also revealed the expression of
NES, NF-L, MAP- 2 and many other neural markers, but in
low expression levels21,41,55. Flow cytometrys, Western
blots, and real time-PCR techniques were used in evaluating
the plasticity. Multipotency of MSCs in vitro showed that
MSCs in culture consistently express native immature neural
proteins such as NES and Tuj-1. Then after induced neural
differentiation, an increase in the expression was occurring
of more mature neural/glial proteins TH, MAP-2, and GFAP
and confirming the ability of MSCs to differentiation into
neuron and astrocyte cells51.
Another study demonstrated that NES, MAP-2, and Tub3
were used as markers to investigate neural differentiation in
the hippocampus of mice after dental pulp-derived stem/
mesenchymal cells implantation which expressed of NES
marker first, then MAP-2 and Tub3 after five days of
differentiation16.
Figure 12: ICC detection results of MSCs after induced differentiation by BME, the results showed increase of NES
protein through different exposure times as revealed under a light microscope, the figures (A-E) represented (MSCs-
29 h) exposure time.
A
B
E
C
D
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Also, there were many other studies which demonstrated the
expression of NES markers first, then other maturation
markers after that, and therefore it was the basic marker in
the detection and determined the early stage of neural
differentiation of MSCs in culture8,52,56, with expression in
both undifferentiated and differentiated MSCs but with
different expression levels.
Assessment efficiency of the current study: The results
showed that the BME was successful to induce neuron cells
formation with higher protein expression through the
differentiation process for the NES (with 61.0, 68.5, 86.3,
61.8% for all exposure time respectively), and NF-L (with
32.2, 65.9, 80.8, 66.4 % for all exposure time respectively)
markers with significant difference level (p< 0.05).
Compared with lower protein expression for MAP-2 marker
(with 4.8, 5.8, 12, 29.7% for all exposure time respectively),
were significant difference levels (p< 0.05) for all exposure
time used in this study. Compared with BHA formula (as the
second differentiation formula) used in this study, it gives
less protein expression with significant difference level at p<
0.05 for all exposure time used in this study as shown in
figure 16. Therefore, the BME was the best differentiation
formula used in the next experiment of regenerative
medicine.
Acknowledgement
This work was supported from the Iraqi Center of Cancer
and Medical Genetic Researches (ICCMGR).
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