Sambucus williamsii induced embryonic stem cells differentiated into neurons
Shih-Ping Liu1,2,3, Chien-Yu Hsu2, Ru-Huei Fu1,4, Yu-Chuen Huang5,6, Shih-Yin Chen5,6, Shinn-Zong Lin1,4,7,8, Woei-Cherng Shyu1,4
1Center for Neuropsychiatry, China Medical University Hospital, Taichung, Taiwan
2Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan
3Department of Social Work, Asia University, Taichung, Taiwan
4Graduate Institute of Immunology, China Medical University, Taichung, Taiwan
5Genetics Center, Department of Medical Research, China Medical University Hospital, Taichung, Taiwan
6Graduate Institute of Chinese Medical Science, College of Chinese Medicine, China Medical University, Taichung, Taiwan
7Department of Neurosurgery, China Medical University Beigan Hospital, Yunlin, Taiwan
8Department of Neurosurgery, Tainan Municipal An-Nan Hospital-China Medical University,
Tainan, Taiwan
Address correspondence to Shih-Ping Liu, PhD, No. 2, Yuh-Der Road, Taichung, Taiwan 40447, Republic of China. Phone: 886-4-2205-2121 ext. 7828; Fax: 886-4-2205-2121 ext. 7810;
Abstract
The pluripotent stem cells, including embryonic stem cells (ESCs), are capable of self-
renewal and differentiation into any cell type, thus making them the focus of many
clinical application studies. However, the efficiency of ESCs differentiated into neurons
needs to improve. In this study, we tried to increase efficiently to a neural fate in the
presence of various transitional Chinese medicines through a three-step differentiation
strategy. From extracts of 10 transitional Chinese medicine candidates, we determined
that Sambucus williamsii (SW) extract triggers the up-regulation of Nestin and Tuj1
(neuron cells markers) gene expression levels. After determining the different
concentrations of SW extract, the number of neurons in the 200 μg/ml SW extract group
was higher than the control, 50, 100, and 400μg/ml SW extract groups. In addition, the
number of neurons in the 200 μg/ml SW extract group was higher and higher after each
time passage (three times). We also detected the Oct4, Sox2 (stem cells markers),
Tuj1, and Nestin genes expression levels by RT-PCR. In the differentiated process,
Oct4 and Sox2 genes decreased while the Tuj1 and Nestin genes expression levels
increased. In summary, we demonstrated that SW could induce pluripotent stem cells
differentiated into neurons. Thus, SW might become a powerful material for neurons–
differentiating strategies .
1. Introduction
Embryonic stem cells (ESCs) have the ability to differentiate into ectodermal,
endodermal, and mesodermal derivatives, giving them significant potential for clinical
cell therapy applications. A lot of neurodegenerative diseases, including Parkinson’s
disease and Alzheimer disease, do not have effective therapeutic methods. Cell therapy
may become a powerful strategy to future clinical applications. However, neurons are
hard to isolate and culture for clinical applications. For this reason, one of the sources to
get neurons for future cell therapeutic applications for neurodegenerative diseases is
ESC. Highly efficient approaches to differentiate ESCs into functional neurons are
critical for modeling neurological disorders and testing potential therapies [1]. Previous
studies have shown that a virus could be used to over-express neural-related genes to
increase the differentiation efficiency [2, 3], but such a method is difficult to use in
clinical applications. Protein transduction-based methods or small compounds can be
efficient, safe alternatives to overcome this challenge [4]. In this study, we tried to
discover if the extract of traditional Chinese medicine could increase the neurons-
differentiation efficiency from ESCs. After we tested ten traditional Chinese medicines,
we found that Sambucus williamsii had the potential to enhance neural-related genes
(Nesin and TuJ1) expression levels. For this reason, we used Sambucus williamsii as
our candidate extract to increase the differentiation efficiency of ESCs.
Sambucus williamsii is widely distributed in the North of China, Korea, and Japan
[5]. In China, it has been used as a medicine for the treatment of bone and joint
diseases for thousands of years [5]. Previous studies have shown that the compounds
isolated from Sambucus williamsii can stimulate effects on osteoblastic UMR106 cell
proliferation [6]. This research also showed that Sambucus williamsii could increase
bone mass and bone strength in ovariectomized rats [7]. However, the correlation
between Sambucus williamsii and stem cells differentiation is unclear.
In this study, we used the ethanol extract of Sambucus williamsii to test the
neurons differentiation ability of ESCs. We used a three-step differentiation strategy to
differentiate ESCs into neurons (Figure 1). The genes expression levels of neural
related genes were determined after Sambucus williamsii treatment by real-time PCR.
After differentiating, immunefluoresce analysis and RT-PCR were used to test the
optimal Sambucus williamsii concentrations for differentiating ESCs into neurons. In the
end, we demonstrated that Sambucus williamsii could increase the neurons- differentiated efficiency of ESCs.
2. Materials and methods
2.1 Mouse embryonic fibroblast (MEF) cultures
Approval for all experimental protocols was applied for and received from the
Institutional Animal Care and Use Committee of China Medical University. MEF isolation
was performed as previously described [8]. Cells were collected from 13.5-day-old
C57BL/6 mouse embryos retrieved by Cesarean section from mice purchased from the
Taiwan National Laboratory Animal Center. Internal organs, legs, and heads were
removed and remaining embryo parts digested with Trypsin. Cells were cultured in
Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat-inactivated fetal
bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 μg/ml), non-essential
amino acids (0.1 mM), and L-glutamine (2 mM) in a humidified incubator (37°C) with 5%
CO2 (all chemicals and solutions from GIBCO BRL). MEF was treated with 10 μg/ml
mitomycin C to become the feeder layers for the stem cell culture.
2.2 Mouse ESC cultures
Mouse ESCs (from the Taiwan Bioresource Collection and Research Center) were
cultured in DMEM with 15% FBS (Hyclone, UT, USA), non-essential amino acids (0.1
mM), L-glutamine (2 mM), β-mercaptoethanol (0.2 mM) (GIBCO BRL), and LIF (4 ng/ml)
(Millipore) in a humidified incubator at 37°C with 5% CO2. 2.3 The strategy of ESC differentiated into neurons
All three-step differentiation procedures were modified from standard protocols
previously described [9, 10] and shown in Figure 1. Briefly, ESCs were transferred to
Ultra-Low attached culture dishes in ES medium without LIF to generate embryoid
bodies (EB). After 4 days, the embryoid bodies were transferred to normal dishes
containing medium with or without the extract of Sambucus williamsii for neurons
selection (P1). After 4 days, cells were subcultured to P2 generation. P3 generation also
followed the same strategy. Immunefluoresces analysis was used for P1, P2, and P3
neurons .
2.4 Real-time PCR and reverse transcription PCR
Total RNA was extracted from ESCs, EB, P1, P2, and P3 neurons using TRIzol
(Invitrogen) [11], with concentrations determined by spectrophotometry. Complementary
DNA was produced from mRNA (5 mg) using a SuperScript III Reverse Transcriptase
Kit (Invitrogen). Real-time PCR was used to determine Nestin and Tuj1 gene expression
levels as previously described [11]. PCR conditions were predenatured at 94°C for 5
min followed by 28 cycles of amplification at 94°C for 30 s, 60°C for 30 s, and 72°C for 1
min, followed by a 10-min extension step at 72°C. PCR was performed with ExTaq
(Takara) to detect the expression levels of the Oct4, Sox2, Tuj1, Nestin and β-actin
genes.
2.5 Immunofluorescent (IF) antibody assays
IF antibody assays were performed as previously described [8]. Cultured cells were
placed on slides, treated with fixing solution I (4% paraformaldehyde plus 400 mM
sucrose in PBS) and held at 37°C for 30 min. Slides were then treated with fixing
solution II (fixing solution I plus 0.5% Triton X-100) and held at room temperature for 15
min. After washing with PBS, slides were treated with a blocking buffer (0.5% BSA in
PBS) at room temperature for 1 h and washed three additional times with PBS prior to
reacting with various primary antibodies (anti-Nestin [abcam] or anti-Tuj1 [GeneTex]) at
1:100 dilutions, either overnight at 4°C or for 1 h at 37°C. Slides were washed five times
with cold PBS before reacting with FITC-conjugated anti-mouse IgG or TRITC-
conjugated anti-rabbit IgG (Sigma-Aldrich). After four more washes with cold PBS,
slides were mounted and observed using a fluorescence microscope. DNA was stained
with DAPI (Sigma-Aldrich) to localize nuclei.
3. Results
3.1 Neural related gene expression levels increased after Sambucus williamsii
treatment
In order to determine that Sambucus williamsii could induce the neural related gene
expression levels and increase the efficiency of ESCs differentiation into neurons, we
measured Nestin and Tuj1 expression levels in the P3 generation after 50, 100, 200 and
400μg/ml Sambucus williamsii treatment. As shown in Figure 2, Nestin expression
levels increased after the 50, 100 and 200μg/ml Sambucus williamsii treatment
compared with the control group, especially in the 50μg/ml. The Tuj1 expression levels
also increased in the 50, 100 and 200μg/ml Sambucus williamsii treatment group
compared with the control group. This data indicates that Sambucus williamsii has the
potential to induce the ESCs toward neuron differentiation.
3.2 Neural related protein expression levels increased after Sambucus williamsii
treatment
After we determined the neural related genes expression levels increased after
Sambucus williamsii treatment, we turned our attention to detecting the real
differentiating ability of Sambucus williamsii. The differentiating strategy shown in Figure
1 and the P3 generation neurons was neural-related protein levels detected by IF
analysis. In Figure 3, the results show that Nestin and Tuj1 protein expression levels in
the 50, 100 and 200μg/ml Sambucus williamsii treatment groups are higher than the
control group and have the dose-response effect. Interestingly, Nestin and Tuj1 protein
expression levels in the 400μg/ml Sambucus williamsii treatment group were not higher
than the control group. Nestin and Tuj1 expression mean the cells were neurons.
Furthermore, this data shows the 200μg/ml Sambucus williamsii treatment to have the
optimal concentration to differentiate ESCs into neurons.
3.3 Neurons differentiation efficiency higher and higher though passage the cells In our differentiating strategy, after passing through EB in normal dishes, cells cultured
in a medium containing Sambucus williamsii passed to the P3 generation. If the
Sambucus williamsii could induce ESCs to neurons, the percentage of neurons should
increase during the passage times. In order to test this hypothesis, we used Nestin and
Tuj1 as the neuron markers to detect the expression levels during the passage times. In
Figure 4, Nestin and Tuj1 expression levels increased from the P1 to the P3 generation
by IF assay. In addition, RT-PCR results also show that Nestin and Tuj1 gene
expression levels increased from the P1 to the P3 generation. The whole stem cell
markers’, Oct4 and Sox2, gene expression levels decreased during the P1 to theP3
generations (Figure 5). These data indicate that the medium containing Sambucus
williamsii leads the ESCs toward neuron differentiation.
4. Discussion
Cell therapy will become a highly potential therapeutic method for many diseases that
do not currently have efficient therapeutic methods, especially neurodegenerative
diseases. However, neurons are hard to obtain and isolate. An efficient strategy to
differentiate ESCs into neurons is very important in future cell therapy. Previous studies
have shown that a virus could be used to over-express neural-related genes to increase
the differentiation efficiency [2, 3] but such a method is hard to use for clinical
applications. Protein transduction-based methods can be efficient, safe alternatives to
overcome this challenge [4]. However, it is difficult for protein to cross cell membranes
and so there is a need to use fusion proteins ( Tat proteins [12-14], for example). Fusion
proteins still have some problems that involve injuring cell membranes. For these
reasons, small molecules may be a better candidate for neurons differentiation.
In this study, we used a three-step differentiation strategy to determine that Sambucus
williamsii could induce ESCs to differentiate into neurons. Most of studies have shown
the application of Sambucus williamsii in bone and osteoblastic research [6, 7]. This is
the first study to investigate the correlation between Sambucus williamsii and stem cells
differentiation. In the end, we are confident we found a new applicationfor Sambucus
williamsii.
In conclusion, this study discovered a traditional Chinese medicine that could
potentially be used to increase the differentiation efficiency of ESCs into neurons. In
addition, a simple method to differentiate ESCs into neurons was also developed. It
should prove helpful in future neurons therapy from the pluripotent stem cells, including
induced pluripotent stem (iPS) cells. We hope it will also be helpful in the future clinical
application of cell therapy.
Acknowledgments
This study is supported in part by the Taiwan Ministry of Health and Welfare Clinical
Trial and Research Center of Excellence (MOHW103-TDU-B-212-113002), and China
Medical University (CMU101-NSC-06). The authors declare no conflicts of interest.
Figure captions
Figure 1. The three-step strategy to differentiate ESCs into neurons.
Figure 2. Nestin and Tuj1 gene expression levels in the P2 generation treated with various concentrations of Sambucus williamsii extract (real-time PCR).
Figure 3. Nestin and Tuj1 expression levels after immunofluorescent staining in the P3 generation treated with various concentrations of Sambucus williamsii extract. Nuclei
were stained with DAPI (blue).
Figure 4. Comparison of Nestin and Tuj1 expression levels after immunofluorescent staining in the P1, P2 and P3 generations treated with various concentrations of
Sambucus williamsii extract. Nuclei were stained with DAPI (blue).
Figure 5. Gene expression levels of Oct4, Sox2, Tuj1, and Nestin in ESCs, EB, P1, P2
and P3 generations as measured by reverse transcription (RT)-PCR.
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