Chapter 4 Role of S1P in human BM- and AD- MSCs proliferation
122

CHAPTER 4 ROLE OF S1P IN HUMAN BM- AND AD- MSCS
PROLIFERATION

In Chapter 3, I discussed the regulatory effects of SPHK inhibitors in human BM- and
AD- MSCs differentiation, switching between the osteogenic and adipogenic pathways.

In this chapter, I present the results obtained in my studies on the role of S1P on the stem
cell proliferation.
S1P has been shown to have proliferative functions, promoting cell growth in various
types of cells (Zhang et al., 1991; Olivera and Spiegel, 1993), including in embryonic
stem cells (Pébay et al. 2005; Harada et al. 2004; Donati et al. 2007). Its structure is
shown below in Figure 4.1.

Figure 4.1 Structure of S1P, D-erythro

As was discussed in Section 1.1 SPHK and S1P, S1P has dual functions. It can act as an
intracellular mediator, and it can also act as an extracellular ligand to bind and stimulate
its-specific receptors, expressed on the same or nearby cells, in an autocrine or a
paracrine manner, to mediate various cellular responses (Spiegel and Milstien, 2003).
What interested us most is S1P role in the stem cells proliferation. So far, there are no
reports on S1P roles in human adult stem cells. Pébay et al. (2005) showed that S1P
could work synergetically with PDGF and promote human embryonic stem cells
Chapter 4 Role of S1P in human BM- and AD- MSCs proliferation
123

proliferation, as well as maintaining their stemness. However, human adult stem cells,
such as MSCs, are very different with embryonic stem cells in terms of cell properties
(such as differentiation potentials) and culture conditions (MSCs attach to the culture

tubes to make 10mM S1P-BSA stock solutions. The stock solution was then diluted with
0.1% fatty-acid free BSA to generate the final working solutions.

4.1.2 Cell Surface Markers Expression (Flow Cytometry)
Human BM- and AD-MSCs (from two different donors) were analyzed by flow
cytometry for their expression of CD29, CD34, CD44, CD 45, CD71, CD73, CD90, and
CD105 using a Guava Personal Cytometer (Guava, Burlingame, Calif., USA). Briefly,
cells were removed from the culture, using 0.125% trypsin and washed once in 2%
FCS/PBS before counting. Cells were rested for 40 minutes in maintenance media before
1x10
5
cells were then aliquoted into a 96-well plate and pelleted at 450 x g for 5 min.
Pre-diluted antibody solutions in 2% FCS/PBS were subsequently added and cells were
incubated on ice for 20 min, followed by two washes in 2% FCS/PBS, before re-
suspension in 100 μl of 4% paraformaldehyde and incubation on ice for 20 min. Cells
were again washed twice in 2% FCS/PBS before being resuspensed in 200 μl of 2%
FCS/PBS and analysed on a GUAVA PCA-96 bench-top flow cytometer (Guava
Technologies Inc., USA). All samples were measured in triplicate for both types of cells.
Mouse IgG1 and IgG2a were used as isotype controls, to determine the positive cell
Chapter 4 Role of S1P in human BM- and AD- MSCs proliferation
125

populations (isotype cells< 5% positive cells). Five thousand events per antibody group
were counted in triplicate samples.

4.1.3 Calcium Assay
Cells were harvested by centrifugation, re-suspended in HERPES calcium (1.5mM)-
supplemented buffer and loaded with 5μM Fura2/AM (Molecular Probes, Invitrogen
Singapore). Human BM- and AD- MSCs were separated into samples of 0.2 million cells,
and were incubated at 37

After that, the membrane was probed with a proper amount of secondary antibody for
two hours, followed by another three 1xPBS washes, each wash lasting five minutes.
Signals were detected by incubating the membrane with SuperSignal
®
West Pico
Chemiluminescent Substrate (PIERCE, # 34080), which is an enhanced
chemiluminescent substrate for detection of HRP, for five minutes and then exposed to a
radiography film (CL-XPosure
TM
Film, PIERCE, # 34090). Alpha-tublin (primary
antibody: Alpha tublin mouse monoclonal antibody (Santa Cruz, #8035); secondary
antibody: anti-mouse IgG HRP (Sigma, A4416)) was used for equal loading control.

4.1.6 Statistical Analysis
Results are expressed as mean ± SD. Significance between mean values was determined
by Student’s t-test. Samples were analyzed by two-sample equal variance, and two-tailed
distribution, with a value of P< 0.05 considered significant.
Chapter 4 Role of S1P in human BM- and AD- MSCs proliferation
127

4.2 RESULTS
4.2.1 Human BM- and AD- MSCs Morphologies
Cell morphologies of human BM- and AD- MSCs under normal culture conditions are
shown in Figure 4.2. Figure 4.2 Morphologies of human BM- and AD- MSCs in the normal culture media.
Human BM-MSCs (A) showed like fibroblast cells, while human AD-MSCs (B) showed
heterogeneous morphologies: some are “flat” , some are “fibroblastic” , and some
are “polar” . The microscopy magnification is 100x.

2
, and S1PR
3
, but less of S1PR
4
and S1PR
5
.

Figure 4.3 S1P receptors expression in human BM- and AD- MSCs. Total RNA was
extracted from both types of cells, and cDNAs for S1PR
1
, S1PR
2
, and S1PR
3
were
prepared as serial controls for real time PCR. Serial controls of S1PR4 and S1PR5 used
in the real time PCR were extracted from human genomic DNA. Results are the average
+ the standard deviation of triplicate samples from at least three separate experiments.
Results for human AD-MSCs represent data from two donors.
S1PRs expression in human BM- and AD- MSCs
0
200

2
2.5
3
day0 10% FBS DMEM 1ul BSA
culture conditions
cell growth (fold)
BM-MSCs
AD-MSCs

Figure 4.4 0.1% FAF-BSA function test in human BM- and AD- MSCs. Human BM-
and AD- MSCs were cultured in different conditions for 4 days, and cell growth was
measured by PicoGreen Assay. Day 0 represents cell numbers at the starting point. Data
from human AD-MSCs represent results from two donors. Results are the average + the
standard deviation of triplicate samples from at least three separate experiments.

4.2.2.3 MSCs growth promoted by S1P
Different dosages of S1P were then tested for their functions in human BM- and AD-
MSCs proliferation. Cells were cultured in serum-free DMEM medium, supplemented
Chapter 4 Role of S1P in human BM- and AD- MSCs proliferation
130

only with different amounts of S1P (100nM, 500nM, 1μM, and 5μM), and cultured for 4
days. The cells growth was measured by PicoGreen Assay, and the results are shown in
Figure 4.5.

Figure 4.5 Function of S1P in human BM- and AD- MSCs proliferation. Human BM-
and AD- MSCs were cultured in different conditions for 4 days, and cell growth was
measured by PicoGreen Assay. Data from human AD-MSCs represent results from two
donors cells. Results are the average + the standard deviation of triplicate samples from at
least three separate experiments. (*P<0.01, vs. “DMEM” in BM-MSCs; **P<0.05, vs.


4.2.2.4 MSCs growth Promoted by S1P and Serum
S1P, together with 10% FBS or 1% FBS, were tested for their roles in MSCs proliferation.
Human BM- and AD- MSCs were seeded in 48-well plates and cultured overnight to
allow for a proper cell adhesion. Cells were washed with 1xPBS and the culture medium
was changed to plain DMEM supplemented with 10% FBS or 1% FBS, with different
amounts of S1P. Cells were cultured for four days and cell proliferation was compared
and summarized in Figures 4.6.
S1P+FBS function in human BM-MSCs proliferation
0%
20%
40%
60%
80%
100%
120%
140%
10%FB
S
10
%
FB
S
+10
0nM

S

60%
80%
100%
120%
140%
160%
1
0%
F
BS
10%FBS+
1
00nM

S
1P
10%FBS
+5
00nM

S
1P
10%FB
S+
1uM
S1P
1%FBS
1%FBS
+
100nM S
Figure 4.6A shows the results from different amounts of S1P, together with 10% or 1%
FBS, on human BM-MSCs proliferation. It is shown that 500nM or 1μM of S1P, together
with 10% FBS, showed to promote about 20% and 23% more cell growth, respectively,
than that cultured with 10% FBS alone. However, only 1μM of S1P together with 1%
FBS, showed to promote around 25% more cell growth, than cells cultured with 1% FBS
only.
Figure 4.6B shows the functions of different amount of S1P combined with 10% or 1%
FBS on human AD-MSCs proliferation. It is shown that cell growth in 500nM S1P+10%
FBS, is 21% more than that cultured in 10% FBS alone; and cell growth in 1μM
S1P+10% FBS shows 30% more than that cultured in 10% FBS alone. Moreover, cells
cultured in 500nM S1P+1% FBS showed 38% more than cells cultured in 1% FBS alone;
and cells cultured in 1μM S1P+1% FBS showed 52% more cell growth than cells
cultured in 1% FBS alone. 100nM S1P did not show to promote cells growth when
combined with 10% FBS, but showed to promote around 30% more cell growth when
combined with 1% FBS.

4.2.2.5 MSCs Growth Promoted by S1P and PDGF-AB
1μM of S1P was combined with 20ng/ml PDGF-AB to investigate their functions in
human BM- and AD- MSCs proliferation. Human BM- and AD- MSCs were cultured for
Chapter 4 Role of S1P in human BM- and AD- MSCs proliferation
133

four days, and human AD-MSCs were also cultured for eight days in different culture
conditions, and the cell proliferation was detected by the PicoGreen assay. The results are
shown in Figure 4.7. Figure 4.7 S1P and PDGF-AB function in human BM- and AD- MSCs proliferation.

**
**
S1P+PDGF-AB function in human AD-MSCs proliferation (8 days)
0
2
4
6
8
10
DMEM DMEM+10%
FBS
DMEM+1uM S1P DMEM+20ng/ml
PDGF-AB
DMEM+1uM
S1P+20ng/ml
PDGF-AB
culture conditions
cells growth (fold)
4.7B
**
**
**
**
Chapter 4 Role of S1P in human BM- and AD- MSCs proliferation
134

It is shown that 1μM of S1P had an additive effect when combined with 20ng/ml PDGF-
AB, in both human BM- and AD- MSCs (Figure 4.7A). In Figure 4.7B, after 8 days
culture, 1μM of S1P and 20ng/ml PDGF-AB promoted around 2-fold increase in cells
growth, compared to the cells grown in DMEM only.

136

In normal culture condition (DMEM+10%FBS), three different cell morphologies were
observed, as was shown in Figure 4.2B and Figure 4.8A. However, in serum-free medium
without any other supplements, most of the cells showed to be elongated, and the cell-cell
adhesion and the cells attachment to the culture flask looked different with the ones
cultured in serum-supplied media. However, 1μM S1P restored the morphology change
observed in human AD-MSCs, and the most obvious difference is that, cells with “flat”
morphology remained in 1μM S1P-supplied culture condition (the arrows shown in
Figure 4.8C), but not in serum-free media (Figure 4.8B).
4.2.3.2 Cell Surface Marker Expression Profile Comparison
Human BM- and AD- MSCs cultured in the serum-supplemented media or S1P-
supplemented media were compared for expression of several typical MSC cell-surface
markers. A series of cell surface markers were tested, and a reasonable hypothesis is that
if along several passages, the cell surface marker expression remained the same, it would
be convincing that the stem cells remained as stem cells.
Starting cells for human BM-MSCs were passage 6, and for human AD-MSCs (cells
from two different donors) were passage 4. Cells were equally grouped and cultured in
four different culture conditions: DMEM, DMEM+1μM S1P, DMEM+10% FBS, and
DMEM+10% FBS+1μM S1P. The cells were analyzed whenever they grew confluent.
However, cells cultured in serum-free DMEM ended up with not enough cells for the
flow cytometry analysis as they almost did not grow, and cells cultured in DMEM+1μM
S1P grew much slower than those from serum-supplied/serum and S1P-supplied ones,
which is consistent with the results we got from Figure 4.5. Since it took them too long
for the cells grew confluent, only the cells from DMEM+10%FBS and
Chapter 4 Role of S1P in human BM- and AD- MSCs proliferation
137

DMEM+10%FBS+1μM S1P culture conditions were analyzed by flow cytometry for
their expression of CD29, CD34, CD44, CD45, CD71, CD73, CD90, and CD105. Cell

CD105
99.66667 99.2 91.73 92.06667 94.13333 92.83333
(B) Human AD-MSCs-donor 1:
Primary
Antibody
% of total events

P=5 P=6 P=7

ctrl S1P ctrl S1P Ctrl S1P
CD29
97.7 95.33333 99.73333 99.53333 99.6 99.63333
CD34
11.43333 6.833333 8.133333 6.733333 14.36667 12.83333
CD44
98.43333 99.26667 99.9 99.83333 99.9 99.76667
CD45
5.233333 2.833333 3.6 3.2 5.833333 3.466667
CD71
8.233333 6.966667 6.633333 4.8 29.63333 16.66667
CD73
90.8 96.33333 99.76667 99.8 99.66667 99.46667
CD90
98.1 97.2 98.63333 98.73333 99.53333 98.86667
CD105
97.23333 96.03333 98.6 97 99.4 99.13333 Chapter 4 Role of S1P in human BM- and AD- MSCs proliferation
138


CD29 CD34
CD44
CD45
Chapter 4 Role of S1P in human BM- and AD- MSCs proliferation
139

CD105
CD29
CD34
Chapter 4 Role of S1P in human BM- and AD- MSCs proliferation
140 Figure 4.10 Cell surface marker expression in human AD-MSCs from the last passage
(P=7). Red solid represents the isotype control, the black line represents the marker

number of the stem cells, only Alizarin Red S Staining was used to quantify the
osteogenic differentiation.
Cells were first seeded in T25 culture flask and cultured overnight to allow for a proper
cell adhesion. On the next day, cells were washed with PBS, and the media was added
that contained the following combination of supplements: DMEM; DMEM+10% FBS;
DMEM+1% FBS; DMEM+1 μM S1P; and DMEM+1% FBS+ 1μM S1P. Culture media
was changed every four days and cells were cultured for 12 days. After that, cells were
counted, and equal amount of cells were seeded in 48-well plates for 24 hours for
Chapter 4 Role of S1P in human BM- and AD- MSCs proliferation
142

adhesion. Cells obtained from the same culture condition were separated into two groups,
a group that did not received the differentiation cocktail, named the uninduced (cultured
in normal growth medium); and a group that received the differentiation cocktail, named
the osteogenic-induced group (cultured in osteogenic differentiation media (section
3.1.1.4
Stem Cells Osteogenic and Adipogenic Induction Culture). Cells were cultured for
a total of 28 days, then fixed and stained with Alizarin Red S. The Alizarin Red S
Staining quantification results are shown in Figure 4.11. Alizarin Red Staining quantification of human AD-MSCs (type1) in different culture
conditions
0
0.05
0.1
0.15
0.2
0.25
0.3

4.11A
Chapter 4 Role of S1P in human BM- and AD- MSCs proliferation
143 Figure 4.11 Alizarin Red S Staining quantifications of human BM- and AD- MSCs in
different culture conditions. Human BM- (A) and AD- (B & C) MSCs were cultured in
DMEM, DMEM+10% FBS, DMEM+1% FBS, DMEM+1μM S1P, or DMEM+1%
FBS+1μM S1P for 12 days. Equal amount of cells from each culture condition were
seeded in 48-well plates with normal culture medium, supplemented with (osteogenic-
induced) or without (uninduced) osteogenic differentiation media for another 28 days.
The cells were stained with Alizarin Red S, and quantified by detecting the absorbance at
500nm. Results are the average + the standard deviation of triplicate samples from at
least three separate experiments. * P<0.05, Student’s t-test.

Figure 4.11A shows the Alizarin Red S Staining quantification in human BM-MSCs
cultured under different conditions. It is shown that the cells cultured from all conditions
showed similar osteogenic differentiation potential. To our surprise, the cells cultured in
DMEM for 12 days did not show to lose the osteogenic differentiation potential.

Figure 4.11B shows the Alizarin Red S Staining quantification in type 1 human AD-
MSCs cultured from different conditions. It is shown that after 12 days, type 1 human
AD-MSCs cultured in DMEM lost about 1/3 of osteogenic differentiation potential as
compared to the cells cultured in DMEM+10% FBS. Cells cultured in the serum-reduced
media (DMEM+1% FBS) showed similar osteogenic differentiation potential with the
Alizarin Red Staining quantification of human AD-MSCs (type2) in different culture
conditions
0
0.05
0.1

4.2.4.1 Intracellular Calcium Release
Calcium is a very important second messenger which can trigger many cellular responses,
including cell proliferation. In this study, intracellular calcium release, in human BM-
and AD- MSCs, was measured when exogenous S1P was added into the cells. Pertusis
toxin (PTX) was used to study the role of S1PRs involvement in S1P triggered cell
proliferation, as PTX is able to bind with Gi subunit of G-protein, and S1PRs are all G-
protein-coupled receptors and shown to bind/activate Gi subunit (Section 1.1 SPHK and
S1P).
Chapter 4 Role of S1P in human BM- and AD- MSCs proliferation
145

Cells were pre-treated or not with 100ng/ml and 300ng/ml of PTX for 30min. After a
stable calcium signal was obtained, 1μM or 10μM of S1P was added into the cuvette.
The calcium release results of human BM- and AD- MSCs at the different treatment
conditions are summarized in Figure 4.12. Figure 4.12 S1P triggers intracellular calcium release in human BM- and AD- MSCs.
Cells were pre-treated with or without 100ng/ml or 300ng/ml PTX for 30min, followed
by adding 1μM or 10μM S1P (the arrow shows). Results were from both BM- and two
donors of AD- MSCs. Experiments were repeated three times.

Chapter 4 Role of S1P in human BM- and AD- MSCs proliferation
146

Figure 4.12 shows, that S1P triggered similar intracellular calcium release in human BM-
(Figure 4.12A) and AD- (Figure 4.12B) MSCs. 1μM S1P triggered a significant rise in
intracellular calcium in both types of cells, and 10μM S1P triggered higher intracellular
calcium levels than 1μM S1P did, demonstrating a dose-dependent effect. 100ng/ml, or
300ng/ml PTX showed to abolish around 80% of the calcium signal triggered by S1P,