Chapter Role of S1P in human BM- and AD- MSCs proliferation CHAPTER 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 122 Chapter Role of S1P in human BM- and AD- MSCs proliferation 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 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) 123 Chapter Role of S1P in human BM- and AD- MSCs proliferation 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 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 resuspension in 100 μl of 4% paraformaldehyde and incubation on ice for 20 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 124 Chapter Role of S1P in human BM- and AD- MSCs proliferation 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 37oC for 30 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 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 and 10 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 125 Chapter Role of S1P in human BM- and AD- MSCs proliferation 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 °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 126 Chapter Role of S1P in human BM- and AD- MSCs proliferation 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 A B 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 127 Chapter Role of S1P in human BM- and AD- MSCs proliferation 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” (flathead 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 copies per 3.5ng of total RNA S1PRs expression in human BM- and AD- MSCs 1400 1200 1000 800 BM-MSCs 600 AD-MSCs 400 200 S1PR1 S1PR2 S1PR3 S1PR4 S1PR5 S1PRs 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 128 Chapter Role of S1P in human BM- and AD- MSCs proliferation 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 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) cell growth (fold) 2.5 BM-MSCs 1.5 AD-MSCs 0.5 day0 10% FBS DMEM 1ul BSA culture conditions Figure 4.4 0.1% FAF-BSA function test in human BM- and AD- MSCs Human BMand AD- MSCs were cultured in different conditions for days, and cell growth was measured by PicoGreen Assay Day 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 ADMSCs proliferation Cells were cultured in serum-free DMEM medium, supplemented 129 Chapter Role of S1P in human BM- and AD- MSCs proliferation only with different amounts of S1P (100nM, 500nM, 1μM, and 5μM), and cultured for days The cells growth was measured by PicoGreen Assay, and the results are shown in Figure 4.5 S1P function on human MSCs proliferation * cell growth (fold) ** BM-MSCs AD-MSCs ** * ** DMEM DMEM+10%FBS DMEM+100nM S1P DMEM+500nM S1P DMEM+1uM S1P DMEM+5uM S1P culture conditions Figure 4.5 Function of S1P in human BM- and AD- MSCs proliferation Human BMand AD- MSCs were cultured in different conditions for 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