Answer Sheets for Reviewer Comments
Comments to the Author
1. In this revised manuscript, the authors failed to address a couple of key issues brought up
previously. The two major conclusions of this manuscript, 1) LTD4 induces migration of ES
cells; and 2) LTD4 promotes proliferation of ES cells, are not convincingly supported by the
experimental data.
 Cell proliferation and migration are simultaneously regulated in many physiological
phenomena. There are many studies including our previous reports about the simultaneous
stimulation of proliferation and migration (1-5). We already demonstrated enough data to
show LTD4-induced migration and proliferation of ES cells in revised manuscript. Combined
with previous and present our observations, we presumed that cell migration appear to partly
contribute to the mechanism of LTD4-induced cell proliferation. If it is needed to define the
precise molecular connections between proliferation and migration of ES cells, further study
is required. This kind of study is out of focus in this experimental object.
*References
(1) Kim et al. (2010). J Cell Physiol. 224(1):187-94.
(2) Suh and Han. (2010). Am J Physiol Cell Physiol. 298(5): C1159-1169.
(3) Park and Han. (2009). Am J Physiol Cell Physiol. 297(4): C935-C944.
(4) Shimizu et al. (2009). Am J Physiol Cell Physiol 297: C389–C396.
(5) Andres et al. (2004). Cardiovasc Res. 63: 11-21.
2. The authors failed to provide convincing evidence that LTD4 could induce migration of
mouse ES cells. Although Figure 6C tried to address this question, the data are indeed
incomplete and misleading. The cell migration images provided to reviewers are very
important data and MUST be included in Figure 6C. Cells in the top row images (control)
exhibited completely different morphology and size from those in the bottom row (LTD4
treated). In fact, only the cells used in the control experiment showed typical mouse ES cell
morphology. If the observed morphological and size differences are due to LTD4 treatment,
the authors should report the result.
 As you indicated, we conducted live cell image analysis for control and inserted the
images in Figure 6C and revised Result section. For live cell imaging, we chose single ES
colony arbitrarily to track the growth and migration in control and experimental group,
respectively. Thus, that is why images had different morphology and size between control and
LTD4. However, in order to avoid confusion as you indicated, we chose representative image
with similar size in revised manuscript as possible and then reanalyzed migration distance.
Please compare each time point image with zero time image, respectively.
180
Control
LTD4
Migration distance (m)
160
140
120
100
80
60
40
20
0
1
2
4
8
10
12
24 (hr)
3. In addition, the authors failed to address if mouse ES cells synchronized by serum
starvation remain undifferentiated. It is no doubt that, after serum starvation, majority of the
cells are in the G1 phase as indicated by Figure 2c, however, the real question is if these cells
are still undifferentiated ES cells. An increase of G1 phase cells after serum starvation could
result from ES cell differentiation rather than synchronization, given the fact that ES cells do
not contain a G1 checkpoint. Figure 1 examined mRNA expression of three pluripotency
related markers and alkaline phosphatase activity; however, it is unclear if cells used in the
experiments were subjected to serum starvation. The legend of Figure 1 in fact indicated the
cells were only treated with LTD4 but not serum starvation. Moreover, the pluripotency
related markers examined in Figure 1 are poorly chosen. Sox2 and Foxd3 are genes essential
for self-renewal of ES cells, yet their expression are not restricted in undifferentiated ES cells.
It is known that level of Oct4 transcript does not significantly decrease even after one week
of EB differentiation (Saxe et al., PLOS One 4:e4467). Therefore, RT-PCR analysis of Foxd3,
Sox2 and Oct4 cannot prove the undifferentiated state of ES cells.
 In our most experimental procedures including Figure 1 clarifying the undifferentiation of
mouse ES cells, ES cells were incubated in DMEM supplemented with LIF and FBS without
a feeder layer for 5 days. The cells were passaged and grown. After 1 - 3 days, the cells were
washed twice with PBS and maintained in serum-free DMEM including all supplements for
24 h. Then, the cells were washed again twice with PBS, incubated with fresh serum-free
DMEM including all supplements, and were treated LTD4 for 12 h before the experiments.
That is, we conducted all experiments under serum-deprived condition to determine the net
effect of LTD4 on ES cells. We described it in detail in the Material and Methods section.
All reports did not say that Foxd3, Sox2 and Oct4 were not profitable for evidences of
undefferentiation of ES cells. In this study, the fact that all these mRNA expression levels
were not decreased was sufficient for regarding that the cells were undifferentiated. Moreover,
as shown below, to determine whether the ES cells differentiated in serum starvation,
differentiation markers such as GATA4 (endoderm), Tbx5 (mesoderm), Fgf5 (ectoderm), and
Cdx2 (trophectoderm) mRNA expression levels were analyzed. However, these marker
expression levels did not change in LTD4 treatment for 5 days. If we cultured ES cells
without LIF, differentiation markers expression levels are increased. Taken together, these
results indicate that ES cells maintained the undifferentiation status under the serum-deprived
condition in this study. In our stem cell research, to determine undifferentiation of ES cells,
we routinely used Foxd3, Sox2 and Oct4 genes expression levels. Indeed, we already
published above 20 papers using this kind of method since 2006. Therefore we have
confidence that these genes expression levels were general markers of undifferentiation status
of mouse embryonic stem cells under our experimental condition (1-7).
GATA 4
Tbx 5
Fgf 5
Cdx 2
Relative to LIF +
5
4
3
2
1
0
LIF -
LIF +
LIF +
LTD 4
* Reference
(1) Lee et al. (2010). J Lipid Res. 2010 Jan 28. [Epub ahead of print]
(2) Suh and Han. (2010). Am J Physiol Cell Physiol. 298(5): C1159-C1169.
(3) Lee et al. (2009). Stem Cells 27(8): 1858-1868.
(4) Yun et al. (2009). Cell Mol Life Sci. 66(9): 1603-1616.
(5) Kim et al. (2008). Stem Cells 26(3): 745-755.
(6) Heo et al. (2007). Stem Cells 25(12): 3069-3080.
(7) Heo et al. (2006). Stem Cells 24:2637-2648