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Introduction
Embryonic stem (ES) cells are
pluripotent cells derived from the inner cell mass of the
pre-implantation blastocyst[1]. These cells can
proliferate in an undifferentiated state and remain totipotent when
grown on a suitable fibroblast feeder layer. Under certain
conditions ES cells are capable of differentiating into a variety of
cell types in vitro, including spontaneously beating cardiac
myocytes[2,3]. The entire differentiating process is rich
in developmental-dependent biological information, for example, the
sequence of expression of cardiac-specific genes and proteins during
differentiation of ES cells in vitro accords with that in
vivo[4]. control of the ES cell mitotic cycle
displays an unusual feature, and regulation of the cell cycle is
critical in maintaining the transition towards differentiation[5,6].
the apoptotic signaling potential of ES cells is necessary to
trigger ES cell differentiation[7,8]. Therefore, the
system of ES cells can provide a platform for studying the effects
of drugs on differentiation and detecting the specific or unique
targets of drug action.
Up to now, the interfering effects
of the traditional chinese medicine on ES cells and their
directional differentiation in vitro have not been
investigated. Icariin (ICA), icaritin (ICT), and desmethylicaritin
(DICT) (Figure 1) are constituents of Epimedium, a
traditional Chinese herbal medicine that has many biological
functions, particularly in cardiovascular function improvement,
hormone regulation, immunological function modulation, and antitumor
activity[9]. Our former work has shown that icariin can
be metabolized to icartin and demethylicartin by human intestinal
bacteria in vitro[10]. Furthermore, icaritin and
desmethylicaritin, but not icariin, exert estrogen-like activity
using the estrogen receptor-positive human breast adenocarcinoma
MCF-7 cell proliferation assay[11]. In addition, icariin
is the major active part in "Xin-shen-ning" tablets, which are used
for the treatment of heart disease[12]. However, the
pharmacological effects and mechanism of the icariin series on the
cardiovascular system are not yet known.
In the present study, the inducible
effects of icariin, icaritin, and desmethylicaritin on the
directional differentiation of ES cells into cardiomyocytes were
examined in vitro. The cardiomyocytes derived from ES cells
were verified using immunocytochemistry. The possible mechanisms of
inducible action in transcription level, cell cycle distribution and
apoptosis were investigated using reverse transcription-polymerase
chain reaction(RT-PCR) and cell cycle analysis, with a view to
detecting the targets of drug action on promoting ES cells to
differentiate in this culture system.
Materials and methods
Animals and cells
NIH mice (male, weighing 24¡À2 g;
female, weighing 22¡À2 g) were obtained from the Experimental animal
Center, Zhejiang University, Hangzhou, China (Grade II, Certificate
No 22-9601018). Mice were housed under 12-h light/12-h dark
and 21¡À1 ºC conditions. To obtain fetuses, mice (3 female and 1
male) were housed together at 17:00. The following morning, when a
copulation plug was detected, was defined as d 0 of gestation. The
permanent ES cell line D3 (ES-D3) was obtained from the American
Type Culture Collection (CRL-1934)[2].
Drugs and reagents Icariin
was obtained from the Drug Biology Product Examination Bureau,
Beijing, China (Batch No 0737-200011, purity 99%); icaritin
was prepared using the cellulose hydrolysis method from icariin, and
icaritin was demethyled using boron tribromide to obtain
desmethyl-icaritin[6]. Purification was carried out by
preparative high-performance liquid chromatography (HPLC) and
identification was conducted using liquid
chromatography/electro-spray ionization mass spectrometry (LC/ESI-MS).
The purities of these compounds were above 98%. retinoic acid (RA),
b-mercaptoethanol, and the antibodies were from Sigma Corproration,
St Louis, Missouri, USA; Dulbecco's modified Eagle's minimal
essential medium (DMEM) and fetal calf serum (FCS) were from Gibco
Invitrogen Corporation, Grand Island, NY USA; the non-essential
amino acids (NEAA) were from Hyclone Logan UT, USA; mitomycin C was
from Kyowa Hakko Kogyo (Tokyo, Japan); and recombinant mouse
leukemia inhibitory factor (LIF) was from Chemicon International
Inc, Temecula, CA, USA. The reagents for the RT-PCR were purchased
from the Shanghai Sangon Biological Engineering and Technological &
Service Company (Shanghai, China).
Cultures of undifferentiated ES
cells fetuses were obtained from the mice on d 13 of gestation
for the preparation of embryonic fibroblast (MEF) cells[13].
The MEF cells of generation three to generation five were used as
feeder cells, which were treated with 1 mg/L mitomycin C for 1 h and
plated at an appropriate density. ES-D3 cells were maintained in an
undifferentiated state by culturing on a monolayer of MEF feeders in
DMEM, supplemented with 10% FCS, 0.1 mmol/L b-mercaptoethanol, NEAA,
and 1¡Á106 U/L LIF.
Differentiation determination of
cardiomyocytes and icariin, icaritin or desmethylicaritin treatment
For differentiation of ES cells, EBs were generated using the
hanging drop method[14,15]. Thirty microlitre of drops
containing approximately 600 ES-D3 cells were placed on the lids of
Petri dishes filled with D-Hanks solution, and cultivated in
hanging drops for 3 d followed by another 2 d in the Petri dishes.
On d 5, EBs were plated separately onto gelatin-coated 24-well
culture plates in differentiation medium that consisted of DMEM, 20%
FCS, 0.1 mmol/L b-mercaptoethanol, and NEAA. icariin, icaritin, or
desmethylicaritin were added to the differentiation medium at a
concentration of 1¡Á10-7 mol/L according to the
preliminary test. Our previous study examining the
concentration-effect relationship of icariin revealed that treatment
with icariin 1¡Á10-7 mol/L significantly
enhanced cardiac differentiation (unpublished data). Thus, a
concentration of 1¡Á10-7 mol/L was used in the
present experiment. ES-D3 cells treated with RA 1¡Á10-8
mol/L and with 0.1% Me2SO were used as positive and
negative controls, respectively.
In the experiment, d 1 referred to
the day of dissociation of ES cells from MEF and the initiation of
differentiation by the formation of EBs. EBs were observed with
light microscopy every day to record the morphology and the number
of the spontaneously beating EBs from d 7 to d 7+23. According to
the percentage of the beating EBs, the inducing effects of icariin,
icaritin, or desmethylicaritin on the directional differentiation of
ES-D3 cells into cardiomyocytes were evaluated using the time-effect
relationship curve. rhythmically beating EBs were considered to be
spontaneously beating cardiomyocytes in EB outgrowths, and were
defined as the marker of successful differentiation[14,15].
Expression of cardiac-specific
proteins analyzed using immunocytochemistry Cardiomyocytes were
isolated from the beating areas of EBs using a modified procedure
described by Maltsev et al[16,17]. For
immunostaining, cells were rinsed twice with PBS, and fixed with
cold acetone for 10 min. After being treated with goat serum for 30
min, specimens were incubated at 4 ºC overnight with the primary
antibody, monoclonal anti-sarcomeric-actinin (clone number EA-53,
1:200 dilution, Sigma) or monoclonal anti-troponin T (clone number
JLT-12, 1:100 dilution, Sigma). The following day, the specimens
were washed with PBS three times and incubated with the fluorescent
antibody: FITC-conjugated F(ab)2 fragment of
affinity-purified goat anti-mouse IgG (1:1000 dilution,
Rockland Inc Gilbertsville, PA, USA) for 1.5 h at 37 ºC. Specimens
were then rinsed in PBS, mounted onto a coverslip with 90% glycerol
in PBS and examined using a fluorescence microscope (Leica
DMIL,California Nevada, Germany).
Expression of cardiac-specific
genes using semi-quantitative RT-PCR The expression of the
cardiac-specific a-myosin heavy chain (a-MHC) gene and myosin light
chain-2v (MLC-2v) in EBs was confirmed using semi-quantitative
RT-PCR. Total RNA was isolated from EBs (n=20) induced by
1¡Á10-7 mol/L icariin, icaritin, or
desmethylicaritin, and 1¡Á10-8 mol/L RA or
solvent, respectively, at d 7+0, d 7+5, d 7+9 using the Trizol
reagent according to the manufacturer's instructions. After
extraction, mRNA was precipitated using the recommended procedures
and dissolved in 0.1% diethylpyrocarbonate solution. To synthesize
first strand cDNA, 7 µL total RNA was incubated in 0.5 µg of oligo (dT)
6 primer and 5 µL deionized water at 65 ºC for 15 min. Reverse
transcription reactions were carried out with 200 U of M-MuLV
reverse transcriptase in 5¡Áreaction buffer and 1 mmol/L dNTP mixture
for 1 h at 42 ºC. Multiplex polymerase chain reactions of 50 µL
contained 1 µL of the RT reaction product, 10¡ÁPCR buffer, 25 U
Taq polymerase, 1 µL of 10 mmol/L dNTP mixtures, and 30 pmol of
each primer. The specific primer pairs were designed as follows: the
cardiac-specific a-MHC gene
(5'-CTGCTGGAGAGGTTATTCCTCG-3',5'-GGAAGAGTGAGCGGCGCATCAAGG-3'; 301 bp)[18]
and MLC-2v
(5'-TGTGGGTCACCTGAGGCTGTGGTTCAG-3',5'-GAAGGCTGACTATGTCCGGGAGATGC-3';
189 bp)[18], and the housekeeping gene b-actin was used
as an internal standard
(5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3',5'-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3';
660 bp).
For the semi-quantitative
determination of a-MHC and MLC-2v mRNA levels, the products of the
reverse transcription reactions were denatured for 3 min at 94 ºC,
followed by 40 cycles (a-MHC), 45 cycles (MLC-2v), and 30 cycles (b-actin)
of amplification in the reaction with Ampli Taq DNA
polymerase: 45 s denaturation at 94 ºC, 40 s annealing at 66.4 ºC
(a-MHC) or 61.1 ºC (MLC-2v) or 55 ºC (b-actin) and 45 s elongation
at 72 ºC. The PCR products were analyzed using 1.5% agarose gel
electrophoresis, visualized with ethidium bromide staining, and
quantified using a bio-imaging analyzer (Bio-Rad, Hercules, CA,
USA), and the density of the products was quantified using Quantity
One version 4.2.2 software (Bio-Rad).
Analysis of cell cycle and
apoptosis by flow cytometry To analyze the cell cycle
distribution of ES cells and apoptosis as parameters in the early
differentiation phase, ES-D3 cells were cultivated without MEF
feeders and LIF, and treated with 1¡Á10-7 mol/L icariin,
icaritin, or desmethyl-icaritin, or 1¡Á10-8 mol/L
RA. After 48 h, the cells were harvested and 1¡Á106
cells were placed into a polypropylene tube and centrifuged at 90¡Ág.
The supernatant was removed and 70% EtOH 1 mL in 4 ºC was dropped
into the cell pellet while vortexing. the cells were kept at 4 ºC
until the DNA was stained. Fixed cells were treated with RNase A in
PBS for 1 h, followed by staining with 50 mg/L propidium iodide in
PBS. Flow cytometric analysis of the cell cycle distribution and
apoptosis was carried out using a BD FACSCalibur with a 488 nm
(blue) argon (Becton Dickinson, San Jose, CA, USA). Data acquisition
was carried out with CellQuest 3.1 software and the data were
analyzed with ModFit LT 3.0 software (Variety Software House,
Topsham, ME, USA).
Statistical analysis Each
data point represents the mean¡ÀSD. At least three independent
experiments were carried out. Statistical significance was evaluated
using one-way ANOVAS with SPSS 10.0 for WINDOWS software. P<0.05
was considered statistically significant.
Results
Apparent cell morphological changes
during the course of differentiation
ES-D3 cells grew aggregates with clear
boundaries and appeared ovoid or nodule shaped on MEF feeder cells
(Figure 2A). Representative EBs at 5 d after the initiation of
differentiation, and just prior to plating onto gelatin-coated
24-well culture plates, were formed by hanging drop cultures. EBs
kept globular shaped structure. (Figure 2B). After plating for 2 d,
cells started outgrowing from the EBs, and cardiomyocytes appeared
as spontaneously contracting cell clusters[14] (Figure
2C). One EB contained one or more beating areas in which the
enlarged size, increased contracting strength, and beating frequency
were observed during the subsequent differentiation phase.
Inducible effect of icariin,
icaritin, and desmethylicaritin on the directional differentiation
of ES-D3 cells into cardiomyocytes Icariin, icaritin, and
desmethylicaritin influenced the degree of cardiac differentiation
of ES-D3 cells. During the course of differentiation (from d 7+2 to
d 7+23), the total percentage of beating EBs treated with 1¡Á10-7
mol/L icariin, icaritin, and desmethylicaritin was 87% (P<0.01),
59% (P< 0.01), and 49%, respectively. In particular, in the
case of icariin at a concentration of 1¡Á10-7 mol/L the
inducing effect on the directional differentiation of ES-D3 cells
into cardio-myocytes was remarkable compared with the control (P<0.05).
Although only 46% of the EBs in control samples contained beating
clusters, 68% of the EBs treated with 1¡Á10-8 mol/L RA
differentiated into beating cardiac clusters (Figure 3A).
The results of the time-effect
relationship revealed that the percentage of differentiation
cultures containing contracting EBs with icariin 1¡Á10-7
mol/L reached a peak level of 85% at d 7+10 and 10% at terminal
stages. The potential of EBs treated with icariin 1¡Á10-7
mol/L undergoing cardiac differentiation was enhanced compared with
control cells over the period from d 7+5 to d 7+23 (P<0.05).
Treatment of EBs with icaritin 1¡Á10-7 mol/L
also resulted in a remarkable increase in the number of EBs with
spontaneously beating cardiomyocytes between d 7+9 and d 7+17 (P<0.05),
whereas the differentiation effect of EBs induced by
desmethylicaritin 1¡Á10-7 mol/L was only
prominent from d 7+9 to d 7+11 (P<0.05) (Figure 3B).
If culture of differentiation was
continued, the numbers of spontaneously beating foci increased and
the spontaneously contractile activity of cardiac myocytes
strengthened[19].
Detection of cardiac-specific
proteins during the differentiation phases of ES-D3 cells into
cardiomyocytes To identify whether the observed cell types
derived from ES-D3 cells expressed cardiac-specific proteins,
indirect immunofluorescence was carried out. The results verified
that differentiated beating cardiac cells stained positively with
anti-a-actinin mAb and anti-troponin T mAb, and these results
support previous studies[20] (Figure 4).
Effect of icariin, icaritin, and
desmethylicaritin on the level of a-cardiac MHC and MLC-2v mRNA
during the early cardiac developmental stage To explore whether
icariin, icaritin, and desmethylicaritin influence the expression
level of a-cardiac MHC and MLC-2v mRNA during cardiomyocyte
differentiation, the beating EBs outgrowths differentiated from
ES-D3 cells were studied using semi-quantitative RT-PCR analysis.
mRNA expression levels of a-MHC by 1¡Á10-7 mol/L icariin,
icaritin, and of MLC-2v by 1¡Á10-7 mol/L
icariin increased during an early cardiac developmental stage for
the period between d 7+0 and d 7+9. In particular, 1¡Á10-7
mol/L icariin-induced MLC-2v expression was detected as
early as d 7+0, whereas increased expression in control cells was
not observed until d 7+5. The experiments also revealed that there
was almost no difference between 1¡Á10-7 mol/L
desmethylicaritin treated cells and control cells from d 7+0 to d
7+9 (Figure 5).
Analysis of cell cycles and
apoptosis using flow cytometry The cell cycle distribution of
ES-D3 cells was considered to be a parameter of their
differentiation state[5,6], which enabled us to determine
the effect of icariin, icaritin, and desmethylicaritin on early
differentiation events before a shift to the cardiomyocyte
phenotype. The results of the cell cycle analysis on propidium-iodide-stained
cells showed a smaller percentage of ES-D3 cells in the G0/G1
phase compared with differentiation cells in control samples (approxi-mately
30% and 40%, respectively). Icariin, icaritin, and desmethylicaritin
1¡Á10-7 mol/L induced the accumulation of cells in G0/G1
(approximately 44%, 40%, and 35%, respec-tively). In particular, the
effect of icariin on the G0/G1 was remarkable
compared with control cells (P<0.05). Treatment of 1¡Á10-8
mol/L RA let ES cells present a similar effect on the
accumulation of cells in G1 (48%). Proportions of S phase
cells in either 1¡Á10-7 mol/L icariin-treated
cells or 1¡Á10-8 mol/L RA-treated cells were
remarkably reduced (29% and 27%, respectively, versus 47% of S phase
in ES-D3 cells; P<0.05) (Figure 6).
Apoptosis plays a vital role in
development by removing unwanted cells[21], which is a
possible checkpoint for the transition towards differentiation of ES
cells. In the present study, ES-D3 cells induced by icariin 1¡Á10-7
mol/L for 48 h showed greater levels of apoptosis (10%)
than control cells (4%) (P<0.05). In contrast, treatment with
icaritin 1¡Á10-7
mol/L or desmethylicaritin resulted in apoptosis levels of
approximately 9% and 8%, respectively. RA 1¡Á10-8 mol/L
treatment for 48 h also led to 8% apoptosis (P<0.05;
Figure 6).
Discussion
ES cell differentiation into
cardiomyocytes in vitro is a unique system that not only
provides opportunities to study cardiomyocyte differentiation, but
also offers a platform for evaluating the effects of drugs and
detecting the specific targets of drug action during cardiogenesis.
In the present study, the possible inducible effects of icariin,
icaritin, and desmethylicaritin on the directional differentiation
of ES cells into cardiomyocytes in vitro were explored. To
investigate the partly inducible effect mechanisms of the three
compounds involved in ES cell differentiation, the expression of
cardiac developmental-dependent genes was detected using RT-PCR.
Cell cycle distribution and apoptosis were analyzed using flow
cytometry.
Our results revealed that treatment
of EBs with icariin resulted in increased and accelerated
differentiation into beating cardiomyocytes. The degree of the
inducible effect of icaritin was less than that of icariin, and
desmethylicaritin had the least inducing effect on the directional
differentiation of ES cells into cardiomyocytes in vitro.
Moreover, cardiac-specific sarcomeric proteins in the cardiomyocytes
derived from ES-D3 cells were identified, since one of the major
questions in muscle development is how a large number of protein
subunits assembles into the remarkably regular structure known as
the sarcomere[20]. Our results confirmed that the
cardiomyocytes were derived from ES-D3 cells.
ES-cell-derived cardiomyocytes
express the cardiac genes in a developmentally controlled manner,
that is, atrial natriuretic factor (ANF), a-MHC, b-MHC, and MLC-2v[4].
In our experiment, a-MHC and MLC-2v were chosen as the evaluation
targets for the directional differentiation. Our experiments
demonstrated that icariin significantly advanced and increased the
mRNA levels of a-MHC and MLC-2v at an early stage of
differentiation, which suggested a partial mechanism for icariin
involving in differentiation could be related to the expression of
the cardiac developmental-dependent genes[4,22, 23].
However, icaritin and desmethylicaritin had less effect on improving
the mRNA level of a-MHC and MLC-2v during cardiac differentiation.
Other studies have shown that a-MHC is expressed relatively early
during the course of cardiogenesis[24]; however, MLC-2v
mRNA is highly expressed relatively late in the process and is
spatially restricted to the ventricular portion[25,26].
Our results support these studies. one of the mechanisms of icariin
in differentiation is to increase and accelerate the mRNA level of
a-MHC and MLC-2v at an early stage of differentiation.
Icariin is a new type of biological
response modifier and differentiation agent[27] and can
induce some cells to differentiate by regulating the cell cycle[27,28].
The cell cycle distribu-tion of ES cells was considered to be a
parameter of their differentiation state[5,6]. This
system of ES cell differentiation could recapitulate the in vivo
differentiation process, including the occurrence of apoptosis
accompanying differentiation. Furthermore, the potential in ES cell
apoptotic signals is necessary to trigger ES cell differentiation.
Alterna-tively, it is possible that some cells enter apoptosis and
the remainder differentiate, and this may depend on their stage in
the cell cycle[7,8]. to elucidate the mechanism of
icariin action on differentiation before a shift to the
cardiomyocyte phenotype, we examined the distribution of the ES cell
cycle and apoptosis using flow cytometry. Our experiment showed that
the induction of icariin was associated with remarkable enrichment
of ES-D3 cells in the G0/G1 phase and a
significant reduction in cells in the S phase. In addition, icariin-mediated
differentiation leads to higher apoptosis. These results suggest
that the effect of icariin is associated with cell cycle arrest and
apoptosis. The balance between positive and negative regulators of
the ES cell cycle is important in maintaining the transition towards
differentiation. The apoptotic signal in ES cells also triggers the
differentiation of ES cells[7].
In conclusion, ES-D3 cells could be
remarkably induced to directionally differentiate into
cardiomyocytes by icariin at a concentration of 1¡Á10-7
mol/L. The promoting effect of icariin on cardiac differentiation
depended on the advancing and increasing gene expression of
a-cardiac MHC and MLC-2v. At an early differentiation stage, the
inducible effect of icariin was related to the regulation of the
cell cycle and the induction of apoptosis.
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