Development of sphingosine kinase (SPHK) inhibitors and the role of sphingolipids in adult stem cell proliferation and differentiation 4

CHAPTER 4 ROLE OF S1P IN HUMAN BM- AND AD- MSCS S1P has been shown to have proliferative functions, promoting cell growth in various types of cells Zhang et al., 1991; Olivera and Spiege

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

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

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 flask while embryonic stem cells are usually cultured on the feeder-layer cells) Therefore,

it is of value to study, whether S1P may promote human BM- and AD- MSCs proliferation, while maintaining their pluripotency

Here, S1P alone, S1P with FBS (10% and 1%), and S1P with PDGF-AB, were tested to investigate their roles in human MSCs proliferation Cell proliferation was measured by Quant-iT PicoGreen assay, which has been introduced in Section 3.1.5 Quantification of the Stem Cell Growth (PicoGreen Assay) All cells used were from passage 2 to passage

9, at which passage they were considered to maintain their multipotency Cells cultured

in S1P-supplemented media were then measured for their stemness maintenance by checking their morphology change, the cell surface markers expression profile, and their osteogenic differentiation potential Finally, signaling pathways that S1P might trigger in human MSCs proliferation were investigated

4.1 MATERIALS AND METHODS

All chemicals, if not specially described, were purchased from Sigma-Aldrich, Singapore

(Some methods used in this chapter are the same with those being used in Chapter 3, which have been addressed in Section 3.1 Materials and methods, in Chapter 3)

4.1.1 Delivery of S1P to Cells

S1P was first dissolved in methanol at 65°C as its solubility in other solvents is very poor

(Van Veldhoven et al., 1989) After S1P was dissolved, specific amounts of S1P were

aliquoted into tubes, and the methanol was evaporated by a stream of nitrogen A suitable amount of 0.1% fat-acid free bovine serum albumin (FAF-BSA) was then added into the 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 1x105 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

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 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 37oC for 30 min and agitated every 15 minutes Afterwards, cells were washed twice and re-suspended in 2ml of HERPES calcium (1.5mM)-supplemented buffer Cells were then placed in a quartz cuvette and placed in a spectrofluorophotometer (Shimadzu RF-5301 PC) Upon getting a stable basal reading, cells were triggered by adding S1P Readings were analyzed by the software provided by the spectro-fluorophotometer manufacturer

(1.5mM)-4.1.4 MAPK (P44/P42) Activation and IκB Phosphorylation

Cells cultured in 6-well plates were washed twice with 1x PBS and starved in serum-free DMEM for half an hour 1μM S1P was then added into the wells and incubated at 37°C for 5min, 10min, 30min, and 60min to look for ERK activation Human AD-MSCs were triggered by 1μM S1P for 5 min and 10 min to look at IkB phsophorylation Incubation was stopped by placing the 6-well plates on ice and the cells were scraped out and collected by centrifugation Cell pellets were lysed and the protein concentrations in the cell lysates were quantified The same amount of proteins from each sample was loaded onto SDS-polyacrylamide (10%) gel electrophoresis (SDS-PAGE) gels and analyzed by

western blotting (more details below) For the primary antibody, phosphor-P44/42 MAPK (Thr202/Tyr204) mouse monoclonal antibody (Cell Signaling, #9106S), and phosphor-IκB-α mouse monoclonal antibody (Cell Signaling, # SC-8404)were used The anti-mouse IgG HRP (Sigma, A4416) was used for the secondary antibody

4.1.5 Western Blotting

The same amount of proteins from the whole cell extracts of each sample were loaded and separated by SDS-PAGE, and the separated proteins were then transferred to PVDF membrane The membrane was probed with a suitable amount of primary antibody overnight at 4 °C, followed by three 1xPBS washes, and each wash lasting five minutes 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-XPosureTM 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

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

A

B

Human BM-MSCs (Figure 4.2A) are more homogenous and look like fibroblast cells Human AD-MSCs (Figure 4.2B) show as a heterogeneous cell population, containing three main types of morphologies: “flat” (round-head arrow shows), “fibroblastic” (flat-head arrow shows), and “polar” (sharp-head arrow shows)

4.2.2 Roles of S1P in human BM- and AD- MSCs

4.2.2.1 S1P Receptors Expression in human BM- and AD- MSCs

As was stated in Section 1.1 SPHK and S1P, S1P functions as an extracellular mediator

are mainly through its five cell surface G-protein-coupled receptors: S1PR1, S1PR2, S1PR3, S1PR4, and S1PR5 Thus, the mRNA expression of these five receptors in human BM- and AD- MSCs was measured by real time PCR, and the results are shown in Figure 4.3 It shows that both human BM- and AD- MSCs, in normal culture condition, expressed higher level of S1PR1, S1PR2, and S1PR3, but less of S1PR4 and S1PR5

Figure 4.3 S1P receptors expression in human BM- and AD- MSCs Total RNA was extracted from both types of cells, and cDNAs for S1PR1, S1PR2, and S1PR3 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

4.2.2.2 BSA effect on the MSCs growth

Since S1P was prepared in 0.1% FAF-BSA, the largest amount of the BSA used in all experiments (1μl) was tested to investigate whether it alone had any effects on the MSCs growth Human BM-MSCs and human AD-MSCs were cultured in different culture conditions (DMEM+10%FBS, DMEM only, and DMEM+1μl of 0.1% FAF-BSA) for 4 days, and the cell growth was detected by measuring the amount of DNA in each sample with PicoGreen assay, which reflected the cell numbers in each sample The results are summarized in Figure 4.4 Cell numbers in the starting day (day 0) was also kept as a control Obviously, the amount of 0.1% FAF-BSA used in our study did not show to promote the stem cells proliferation

BSA function test in human MSCs (4 days)

Figure 4.4 0.1% FAF-BSA function test in human and AD- MSCs Human 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

BM-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

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 and AD- MSCs proliferation Human 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

BM-“DMEM” in AD-MSCs Student’s t-test)

Figure 4.5 shows that S1P alone, together with DMEM, showed to promote the cells proliferation in human BM-MSCs only when the cells were cultured in DMEM+1μM S1P Moreover, 500nM and 1μM of S1P promoted around 1.2-fold and 1.3-fold of human AD-MSCs growth, respectively, as compared with the cells growth in DMEM alone Additionally, it is shown that a higher amount of S1P (5μM) did not show to promote human AD-MSCs proliferation

S1P function on human MSCs proliferation

* **

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

S1P

10%F

BS+500nM

S1P

10%FBS+

1uM

S1P

1%FBS

1%FBS+100

nM S1P

1%FBS+500

nM S1P

1%FBS+1uM

Figure 4.6 Function of S1P and serum 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<0.05, vs 10% FBS; **P<0.05, vs

“1% FBS Student’s t-test)

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

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 Human BM- and AD- MSCs were cultured in different culture conditions and cells proliferation were tested by PicoGreen assay Results are the average + the standard deviation of triplicate samples from at least three separate experiments (*P<0.05, vs DMEM in human BM-MSCs; **P<0.05, vs DMEM in human AD-MSCs Student’s t-test)

S1P+PDGF-AB function in human MSCs proliferation (4 days)

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

4.2.3 Stemness Maintenance of Cells Cultured with S1P

After identifying S1P functions (S1P alone, S1P with serum, S1P with PDGF-AB) on human BM- and AD- MSC proliferation, cells cultured in S1P-supplemented media were tested for their stemness maintenance

In particular, cells cultured from serum-supplemented or S1P-supplemented media were compared for their morphologies, their cell surface markers expression profile, and their osteogenic differentiation potential It should be noted that only osteogenic differentiation potential was selected as a “proof-of-principle” for the MSCs multipotency test

4.2.3.1 Cell Morphologies Comparison

In most of the experiments, cells cultured in serum-free media seemed to be “elongated” compared with those cultured in serum-supplemented media However, the presence of S1P partially restored the cell morphology observed in serum-free culture conditions This finding was observed for both human BM- and AD- MSCs, but more obvious in human AD-MSCs The results are shown in Figure 4.8

Figure 4.8 Human AD-MSCs morphologies in different culture conditions Cells were cultured from DMEM+10%FBS (A); DMEM (B); and DMEM+1μM S1P (C) Magnificence: 120x (the arrows show the cells morphology as “flat”)

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 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

S1P-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