Document Type : Original Article
Authors
1 Department of Anatomy, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran
2 Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
3 Department of Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
Abstract
Keywords
Introduction
Primordial germ cells (PGCs) are derived from the
extreme proximal region of the epiblast adjacent
to the extra-embryonic ectoderm. Some growth
factors have been shown to induce PGCs differentiation
from stem cells (
BMPs have known pivotal roles in germ cell
development and function, in particular BMP4
controls the formation and early proliferation of
PGCs. BMP4, which is released from extra-embryonic
ectoderm, provides a proper condition for
PGCs differentiation from epiblast cells (
Some reports exist regarding the differentiation of
PGCs from embryonic stem cells (ESCs) by inducing
formation of embryoid bodies (EBs) and the
addition of certain growth factors, such as BMP4,
to cultures. Several studies have demonstrated the
ability of EBs to support differentiation of germ cells
Recently, the development of PGCs derived from
epiblast cells using an
Despite previous reports that have shown which
BMP4 in tissue culture acts in a dose dependent
manner, few studies have demonstrated the effects
of functional doses of BMP4 on PGCs differentiation
from mouse embryonic stem cells (mESCs)
This project was approved by the Ethics Committee of Tehran University of Medical Sciences. In this study, we utilized concentrations of 0, 5 and 10 ng/ml of BMP4 (R & D System, USA) for a culture period of two days. We designed our control and treatment groups based on the receiving doses of BMP4 as follows: D2B0 for the control group, D2B5 and D2B10 for treatment groups.
CGR8-GFP mESC, established from strain 129 (a
gift from Dr. M. Solimani), was maintained in the
absence of feeder cells. CGR8-GFP was cultured
on gelatin (0.1% Sigma, USA)-coated 50 ml plastic
flasks (Falcon, Becton Dickinson) in knock out
dulbecco’s modified eagle medium (DMEM) that
contained high glucose and pyruvate (Gibco-Life
Technologies, Canada) supplemented with 10% fetal
bovine serum (FBS); (Gibco-Life Technologies, Canada,
batch no. 12021565), leukemia inhibitory factor
(LIF) (1,000 IU/ml; Chemicon, Boronia, Australia),
1% w/w non-essential amino acids (Gibco-Life
Technologies, Canada), 0.1 mM β-mercaptoethanol
(Sigma, USA) and 1% w/w penicillin/streptomycin
(Gibco-Life Technologies, Canada). The cells were
incubated at 37°C in 5% CO2. Primary cultures were
allowed to reach confluence before cells were lifted,
split and replated (
EBs were created using the hanging drop method.
Once secondary ESC cultures reached confluence,
cells were lifted as described before, washed and
resuspended in LIF-free knock out DMEM supplemented
with 10% FBS to a concentration of
2000 cells per 20 μl. Twenty micro liter drops of
the suspension were placed on the lid of a 10 cm
plastic culture dish (Falcon). The lid was turned
upside down and placed on the bottom part of
dish, which was filled with sterile water, creating
hanging drops. The cells were incubated at 37°C
in 5% CO2. EBs were cultured for 48 hours before
being transferred to differentiation medium for the
a period of two days (
For immunocytochemistry, EB cells in each
group were washed with phosphate buffered saline
(PBS) at pH 7.4 and fixed in 4% paraformaldehyde
(PFA) for 30 minutes at room temperature
(RT). Fixed cells were permeabilized with
0.2% triton X-100 for 10 minutes at RT followed
by three washes with PBS. To block unspecific
binding of the antibody, cells were incubated with
10% goat serum for 30 minutes at RT. Then, cells
were incubated with primary antibody
EB cells were washed with PBS and treated
with trypsin/EDTA for 5 minutes to form single cells. The suspension was collected by centrifuging
at 2000 rpm for 5 minutes. For intracellular
staining of
EB cells were collected at the designated periods of the culture by aspiration into an Eppendorf tube, centrifuged, and the supernatant discarded. Total RNA was extracted using the qiazol lysis reagent (Qiagen) according to the manufacturer’s instructions. Total RNA was quantified and 5 μg was used for cDNA synthesis using random primers (Fermentas) under standard conditions. RT-PCR amplifications were conducted for 3 minutes at 95°C (95°C, 30 seconds; 60°C, 45 seconds; and 72°C, 45 seconds) for 40 cycles and 72°C for 7 minutes for the final extension. Primer sequences are shown in table 1.
Quantitative RT-PCR primer sequences
Gene | Primer (forward/reverse) | Significance |
---|---|---|
5'-CTTCTTGGGTATGGAATCCTG -3' 5'-GTGTTGGCATAGAGGTCTTTAC. -3' | Internal control | |
5'-GTTCTCTTTGGAAAGGTGTTC-3' 5'-GCATATCTCCTGAAGGTTCTC -3' | Pluripotency marker | |
5'- TGAAGAGGACGCTTTGGA-3' 5'- CTTTCAGCACCGACAACA -3' | Germ cell marker | |
5'-CGGAGAGGAACCTGAAGC -3' 5'- CGCCAATATCTGATGAAGC -3' | Germ cell marker | |
Quantitative data were expressed as means ± SEM from at least three experiments. One-way ANOVA was used for statistical analysis with p<0.05 considered significant.
In this study, we assayed PGCs formation from
CGR8-GFP and
Immunocytochemistry staining of EB cells for fluorescent cells. First panel: D2B0 (A), D2B5 (E) and D2B10 (I). The second panel shows ICC staining of EB cells for Mvh: D2B0 (B), D2B5 (F) and D2B10 (J). The third panel shows DAPI staining; D2B0 (C), D2B5 (G) and D2B10 (K). Scale bars: 50 μm. Analysis by flow cytometry indicated a small population of Mvh-positive cells in mESC. Proportion of PGCs derived from mESC in D2B0 (0.32%) (D), D2B5 (7.63%) (H) and D2B10 (9.54%) groups (L), Negative controls are shown in black.
In the control (undifferentiated) group less than
0.5% of the population was positive for
To recognize other specific characteristics of PGCs
derived from mESC, we applied immunocytochemistry.
Among the entire marker that was used
to distinguish between mESCs and PGCs,
Expressions of
Reverse transcription–polymerase chain reaction for the identification of germ cell markers. (A) RNA was prepared from the control group (D2B0) for Oct4, Stella and Mvh. (B) RNA was prepared from treated groups (D2B5 and D2B10) for Oct4. RNA was prepared from treated groups (D2B5 and D2B10) for Stella and Mvh. β-actin served as an internal mRNA control.
Another gene,
BMP4, a mesoderm inducer, plays an important
role in PGC generation in vivo (
An EB differentiation process was firstly adopted
because a 3-d culture would more closely mimic
conditions in situ than a monolayer culture (
The percent of
The ability of exogenous BMP4 to potentiate germ
cell differentiation has been previously evaluated.
In this study we demonstrated that BMP4
was sufficient to form PGCs in an
Epiblast cells could be differentiated into PGCs in
the presence of recombinant human BMP4. The
ratio of PGCs derived from epiblast cell culture increased
with BMP4 (
Previous studies have demonstrated that there are
Vasa-positive and Vasa-negative cells in EBs, but
the higher percentage belongs to Vasa-negative
cells. Thus, some of these cells were either undifferentiated
or differentiated to other cells (
Thus, perhaps, only a small proportion of cells in
differentiating human EBs express receptors required
for establishment and/or maintenance of
PGCs in ES cells (
Also Vasa-positive cells were most frequently
localized to the edge of EBs (
Taken together, these results indicate that BMP4
triggers PGCs derivation by providing a favorable
microenvironment in an