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

Chapter Introduction CHAPTER INTRODUCTION Sphingolipids, components of membrane lipids, have emerged as the sources of several important signalling molecules (Tay et al., 2005) Sphingolipids are a class of lipids derived from the aliphatic amino alcohol sphingosine (Figure 1.1) Sphingolipids, such as ceramide and sphingosine-1-phosphate (S1P), belong to a new class of potent bioactive molecules; these sphingolipids have been shown to be involved in a variety of cellular processes, including cell differentiation, apoptosis and proliferation (Spiegel and Merrill, 1996; Hannun et al., 1994; Heller et al., 1994; Kolesnick and Golde, 1994; Wang et al., 1996; Geoffroy et al., 2004) Figure 1.1 Structure of Sphingosine, D-erythro Sphingomyelin, the major membrane sphingolipid, is the precursor of the bioactive ceramide, sphingosine and S1P When sphingomyelin is hydrolyzed by sphingomyelinases, ceramide is formed; ceramide can then be hydrolyzed by ceramidases to produce sphingosine, and sphingosine in turn can be phosphorylated, by sphingosine kinases (SPHKs), to yield S1P; this metabolic process is summarised in Figure 1.2 Ceramide and sphingosine are implicated in diverse stress-related responses, such as cellcycle arrest and apoptosis (Kolesnick and Golde, 1994; Hannun et al., 1996; Spiegel and Merrill, 1996) In contrast, S1P has been shown to regulate cell growth (Zhang et al., 1991; Olivera and Spiegel, 1993) and suppress programmed cell death (Cuvillier et al., Chapter Introduction 1996 and 1998; Edsall et al., 1997) It has been suggested that the balance between the intracellular levels of ceramide and S1P could determine the cell fate (Cuvillier et al., 1998; Morita et al., 2000; Perez et al., 1997; Xia et al., 1999) Figure 1.2 The sphingolipid metabolic pathway Sphingomyelin is hydrolyzed by sphingomyelinases to form ceramide Ceramide is metabolized by ceramidase to generate sphingosine SPHK phosphorylates sphingosine into S1P, which is further cleaved by S1P lyase to a fatty aldehyde and ethanolamine phosphates 1.1 SPHK AND S1P So far, two mammalian SPHKs have been cloned, sequenced and characterized These kinases are encoded by two genes, SPHK1 (Kohama et al., 1998; Melendez et al., 2000; Pitson et al., 2000), and SPHK2 (Liu et al., 2000) Comparison of the two isoforms of SPHKs is shown in Table 1.1 (modified from Liu et al., 2002) Both SPHKs possess a conserved kinase catalytic domain which contains the ATP-binding site, as well as five other conserved domains (Liu et al., 2002; Pitson et al., 2002) These domains may play a role in substrate recognition (Pitson et al., 2002) Both SPHK1 and SPHK2 are capable of phosphorylating erythro-sphingosine, dihydrosphingosine and phytosphingosine; however, no other phospholipids appear to be significantly phosphorylated by these enzymes (Kohama et al., 1998; Melendez et al., 2000; Pitson et al., 2000) Despite the overall homology of the conserved domains and substrate recognition of SPHK1 and SPHK2, diversity between the gene sequences implies they did not arise from a simple gene-duplication event (Liu et al., 2002) Chapter Introduction Moreover, SPHK1 and SPHK2 have been shown to possess different kinetic properties (SPHK1>SPHK2) and different temporal expression patterns during development (Spiegel and Milstien, 2003) Therefore, it is reasonable to deduce that these two isoforms may have distinct cellular functions and may be regulated by different signalling mechanisms Unfortunately, so far, no 3-dimention structure information is available for SPHK1 and SPHK2, which, to a certain extent, limits functional studies on these two isoforms Table 1.1 SPHK1 MW(kDa) 42 Number of AA 384 Tissue expression Lung, spleen>>kidney, (mouse) liver, brain Membrane-association 17% Inhibitor DMS (competitive) DHS (competitive) Comparison of SPHK1 and SPHK2 SPHK2 66 618 Liver, heart>>kidney, brain, testes 20% DMS (noncompetitive) SPHK activity has been shown to be stimulated by several external stimuli including growth factors such as, platelet derived growth factor (PDGF) (Olivera et al., 1999), nerve growth factor (NGF) (Edsall et al., 1997); cytokines such as tumor necrosis factorα (TNFα) (Xia et al., 1999; Liang et al, 2005); phorbol esters (Olivera et al., 1993), antigen receptors such as IgG and IgE receptors (Melendez et al., 1998, 2002); and several other receptors/stimuli (reviewed in Tay et al 2005) All those diverse external stimuli can activate the endogenous SPHK to generate S1P S1P has been reported to possess dual functions (Spiegel and Milstien, 2003) It was initially suggested as an intracellular second messenger as some growth factors, like PDGF, NGF and TNFα-, could activate SPHK and increase S1P level in the cells Chapter Introduction (Olivera and Spiegel, 1993) In addition, some reports showed that intracellular S1P can induce cell proliferation and survival (Desai et al., 1992), as well as calcium mobilization (Zhang et al., 1991; Ghosh et al., 1990) However, no potential intracellular receptor(s) that mediate S1P intracellular functions have been identified so far (Kluk et al., 2002) More recently, researchers found that S1P could function as an extracellular mediator by stimulating S1P receptors, present on the cell surface of the same or nearby cells, in an autocrine or a paracrine manner (Spiegel and Milstien, 2003) S1P receptors (S1PRs) are members of the endothelial differentiation gene (EDG) G-protein-coupled family of receptors So far, EDG1/S1PR1, EDG5/S1PR2, EDG3/S1PR3, EDG6/S1PR4, and EDG8/S1PR5, five members have been identified (Chun et al., 2002) These five receptors are coupled to different G proteins (for example, S1PR1 and S1PR4 are coupled mainly to Gi; S1PR2 and S1PR3 activate Gi, Gq and G12/13; and S1PR5 is coupled to Gi and G12/13 (Spiegel and Milstien, 2003) Among all five S1PRs, S1PR1, S1PR2 and S1PR3 are most widely expressed, while S1PR4 is mainly found in hematopoietic system, and S1PR5 is predominantly expressed in brain and spleen (MacLennan et al., 1994; McGiffert et al., 2002; Okazaki et al., 1993; Yamaguchi et al., 1996; Zhang et al., 1999; Liu et al., 2000; Kluk and Hla, 2002) Interaction of S1P with these receptors regulates different cellular processes such as migration, proliferation, cytoskeletal organization, adherens-junction assembly and morphogenesis (Kluk and Hla, 2002) in vitro; as well as other physiological processes such as blood vessel maturation, cardiac development and angiogenesis in vivo (Liu et al., 2000; Ishii et al., 2002) In particular, S1PR1 showed to affect cell survival/proliferation, migration, cytoskeletal organization (Kluk and Hla, 2002), and it is necessary for vascular Chapter Introduction maturation in vivo (Liu et al., 2000) S1PR2 was reported to regulate the role of S1P in heart cell migration during embryogenesis in zebrafish (Ishii et al., 2002), indicating its function in cardiovascular system Interestingly, while S1PR1 has been shown to promote cell survival/proliferation, S1PR2 has been shown to activate stress-associated kinases leading to apoptosis (Kluk and Hla, 2002) S1PR3 was found to share most of the signaling aspects of both S1PR1 and S1PR2, and its functions can be substituted by other S1PRs (Kluk and Hla, 2002) Compared to the other three S1PRs, much less is known for the function of S1PR4 and S1PR5 It was found that S1PR4 could mediate activation of adenylyl cyclase in response to low doses of S1P in mouse embryonic fibroblasts (Ishii et al., 2001) Wang et al (2005) reported that S1PR4 could mediate immunosuppressive effects of S1P by inhibiting proliferation and secretion of cytokines, while enhancing secretion of the suppressive cytokine Interleukin-10 (IL-10) S1PR5 is the most recent member in S1PRs family and its functions in vitro and in vivo still remain to be determined There are some reports suggesting that S1PR5 could inhibit adenylyl cyclase in a pertussis toxin (PTX)-sensitive manner (Im et al., 2000; Malek et al., 2001) Jaillard et al (2005) reported that S1PR5 is an oligodendroglial receptor with dual functions on process retraction and cell survival It is also interesting to note that S1P, binding to S1PR5, was reported to inhibit serum-induced extracellular signal-regulated kinase (ERK) and activation (Malek et al 2001), suggesting that stimulation of S1PR5 might have anti-proliferative effects A brief description for S1P intracellular and extracellular functions is shown in Figure 1.3 (modified from Spiegel and Milstien, 2003) Chapter Introduction Stimulus Plasma membrane Survival Downstream signaling Growth SPHK Ca2+ Cellular responses Sphingosine S1P Downstream signaling G S1P S1PRs Figure 1.3 S1P signaling as a dual functional factor Various stimuli could activate endogenous SPHK SPHK would phosphorylate its substrate-sphingosine, into S1P S1P has dual functions On the one hand, it could act as an intracellular mediator and trigger numerous cellular events, including intracellular calcium release, and promote cell survival and cell growth On the other hand, it could be secreted and function in an autocrine or a paracrine fashion, to bind with its G-protein-coupled receptors (S1PRs), and trigger other downstream signaling pathways More information about SPHK and S1P functions and applications in some pathological processes and in stem cell research will be addressed in the following sections 1.2 SPHK AND S1P REGULATION IN PATHOLOGICAL STATES SPHK and S1P have been suggested to be potentially involved in several pathological diseases, including in inflammatory diseases and cancer 1.2.1 SPHK and S1P in Inflammation The activation of SPHK could exert a proinflammatory effects by promoting neutrophil chemotaxis (Cummings et al., 2002; Ibrahim et al., 2004), and induced certain proteins Chapter Introduction important in inflammation, such as cyclooxygenase-2 and monocyte chemoattractant protein-1 (Pettus et al., 2003; Wu et al., 2004; Chen et al., 2004) Moreover, SPHK is required for antigen-receptors, on mast cell and monocytes, to trigger acute inflammatory responses (Choi et al., 1996; Melendez et al., 1998 and 2002; Jolly et al., 2004) Additionally, S1P has been shown to induce eosinophil chemotaxis (Roviezzo et al., 2004) Therefore, the stimulatory effect of SPHK and its product S1P on immune cells such as monocytes, neutrophils, mast cells and eosinophils, suggest that SPHK and its product S1P may play key roles in inflammation In humans, the gene for SPHK1 maps to a region on chromosome 17q (Melendez et al., 2000), which contains several genes involved in autoimmune diseases, including in multiple sclerosis (Kuokkanen et al., 1997), psoriasis (Nair et al., 1977), and epidermodysplasia verruciformis (Enlund et al., 1999) Therefore targeting SPHK and/or S1P may have profound therapeutic applications Indeed, recently a novel drug (FTY720, “fingolimod”), a structural analogue of sphingosine, is undergoing clinical trials as a novel therapeutic to treat autoimmune diseases (Mansoor and Melendez, 2008) FTY720 could be phosphorylated by SPHKs (Brinkmann et al., 2002; Mandala et al., 2002; Paugh et al., 2003; Billich et al., 2003) and the phosphorylated form of FTY720, FTY720P, is an agonist of all S1P receptors except S1PR2 (Brinkmann et al., 2002; Mandala et al., 2002; Taha et al., 2006) By preventing S1P binding to S1PRs, FTY-720P induced an increase in lymphocyte homing from the blood to peripheral lymph nodes and peyers patches, as well as an inhibition of lymphocyte egress from the thymus (Yagi et al., 2000) and lymph nodes into the bloodstream (Chiba et al., 1998) In this way, FTY-720P elicited blood lyphopenia Chapter Introduction FTY720 is currently in phase III clinical trials as a mono-therapy for relapsingremitting multiple sclerosis (ClinicalTrials.gov 2007-08-20) The way FTY-720P works to suppress S1P implies that immune response triggered by activated SPHK might be, at least in part, due to S1P functions through its membrane receptors, which suggests that inhibiting SPHK activity and/or S1P-receptors could be novel therapeutic strategies for treating inflammatory diseases 1.2.2 SPHK and S1P in Cancer SPHK1 has been reported to express in higher level in tumor tissues when compared to normal tissues (Hong et al., 1999, Xia et al., 2000) Moreover, it has been proposed that SPHK1, if up-regulated, could act as an oncogene (Xia et al., 200) Furthermore, it had been found that the inhibition of SPHK is anti-proliferative and pro-apoptotic for melanoma cells (French et al., 2003) It has been suggested, that the involvement of SPHK and S1P in cancer diseases is partially due to their roles on tumor-associated angiogenesis (Argraves et al., 2004; Hla, 2004; Taha et al., 2006), and partially due to their proliferative roles on the tumor cells themselves (Ogretmen and Hannun, 2004) S1P could function through S1PRs on the endothelial cell surface membrane and regulate endothelial cell survival (Limaye et al., 2005), migration (Kimura et al., 2000; Lee et al., 2001), barrier enhancement (Schaphorst et al., 2003), and blood vessel stabilization via interactions with mural cells (a process requiring N-cadherin) (Paik et al., 2004) All these findings support the proposition that S1P is closely involved in new blood vessel formation, which is a critical process in tumour establishment and growth Chapter Introduction Very recently, a monoclonal antibody against S1P has been developed and shown to bind and neutralize extracellular S1P, at its physiologically relevant concentrations (Visentin et al., 2006) Moreover, this monoclonal was shown to have potentially therapeutic usage in reducing tumour growth, invasion, and vessel formation in multiple murine models (Visentin et al., 2006) Taken together, all these reports strongly suggest that SPHK1 and S1P are potential novel targets for cancer therapy 1.2.3 SPHK and S1P in Other Diseases Several groups are providing evidence for a role for SPHK activation in cardiovascular and metabolic pathogenesis, such as atherosclerosis and diabetes The role of SPHK and S1P in atherogenesis is still controversial, as some studies imply that the S1P may protect against atherosclerosis (Kimura et al., 2001; Nofer et al., 2004), while others indicate that S1P may be involved in the onset and/or development of atherosclerosis (Xia et al., 1998; Auge et al., 2000; Siess et al., 2000; Taha et al., 2006) S1P has been found to form a complex with high-density lipoproteins (HDL), and lowdensity lipoproteins (LDL); with HDL containing more S1P than LDL and very lowdensity lipoproteins (VLDL) (Xu et al., 2004) Oxidized LDL is a major risk factor for atherosclerosis, and it can sequentially induce sphingomyelinase, ceramidase and SPHK in smooth muscle cells, resulting in S1P production and enhanced mitogenesis of these cells (Auge et al., 1999) Other growth factors, such as basic fibroblast growth factor (bFGF), have been shown to induce hyper-proliferation by activating SPHK (Xu et al., 2002) Xia et al (1998) reported that in endothelial cells, TNFα induced ERK and Chapter Introduction nuclear factor κB (NF-κB) activation through SPHK activation, while HDL could inhibit all of these, by inhibiting the SPHK activity triggered by TNFα These findings suggested an atherogenic role for SPHK activation Interestingly, Nofer et al (2004) reported that S1P functioned as an anti-atherogenic, hypotensive, and vasoprotective molecule On the other hand, Deutschman et al (2003) reported that S1P appeared to be more predictive indicative of atherogenesis in clinical trials than many other well-established risk factors, indicating that the levels of serum S1P correlate with the severity of the disease This would indeed suggest that SPHK/S1P could be a potential therapeutic target for atherosclerosis The hyper-proliferative role of SPHK and S1P has been proposed to contribute to the early stages of diabetic nephropathy (Katsuma et al., 2002 and 2003; Geoffroy et al., 2004) In recent reports, streptozotocin-induced diabetes enhanced neutral ceramidase and SPHK activities, which resulted in increased mesangial proliferation, key events in the pathogenesis of diabetes (Katsuma et al 2002; 2003; Geoffroy et al 2004) In summary, SPHK and S1P appear to be involved in various pathological processes SPHK activation generates S1P, which can function as an intracellular mediator, as well as an extracellular mediator by binding to its receptors to stimulate various downstream signaling pathways, to regulate cell survival, cell proliferation and migration Moreover, these processes are key events in the various pathological conditions, as discussed above Furthermore, the literature discussed also indicates that SPHK and/or S1P are involved in inflammatory diseases, cancer, atherosclerosis and diabetes 10 Chapter Introduction 1.4.4 Clinical Potentials of MSCs MSCs possess the capability of differentiating into a wide variety of sub-populations, such as: adipose (Pittenger et al., 1999; Zuk et al., 2001 and 2002; Rodriguez et al., 2005a; Mauney et al., 2005; Song et al., 2007; Wall et al., 2007), bone (Conget et al., 1999; Zuk et al., 2001 and 2002; Reyes et al., 2001; Cowan et al., 2004; Huang et al., 2002; Hicok et al., 2004), cartilage (Pittenger et al., 1999; Reyes et al., 2001; Sekiya et al., 2002; Erickson et al., 2002), cardiac muscle (Rangappa et al., 2003; Gaustad et al., 2004; Planat-Benard et al., 2004a; Fukuda et al., 2005; Vilquin et al., 2006; Zhang et al., 2006; Djouad et al., 2006; Cipriani et al., 2007; Yang et al., 2007), endothelial cells (Miranville et al., 2004; Yang et al., 2007), hematopoietic cells (Cousin et al., 2003; Drouet et al., 2005;), hepatocytes (Shu et al., 2004; Sato et al., 2005), neuronal cells (Ashijian et al., 2003; Safford et al., 2004; Tao et al., 2005; Kim et al., 2005; Tropel et al., 2006; Chen et al., 2006; Benvenuti et al., 2006; Yim et al., 2007), and skeletal muscle (Young et al., 2001; Mizuno et al., 2002; Bacou et al., 2004; Santa Maria et al., 2004; Krampera et al., 2006) Thus, MSCs have been suggested to have therapeutic potentials in the treatment of osteoporosis, osteogenesis imperfecta and osteoarthritis (Kumar et al., 2004; Bielby et al., 2007; Wang et al., 2006; Gimble et al., 2006; Nirmalanandhan et al., 2007; Giordano et al., 2007; Luyten 2004), muscular disorders (Mackenzie et al., 2001; Peng et al., 2003;), nervous system disorders (Phinney et al., 2005; Safford et al., 2005; Isakova et al., 2006; Uccelli et al., 2006;) immune disorders (Uccelli et al., 2006; Corcione et al., 2006), and heart failures (Ohnishi et al., 2007; Behfar et al., 2007) Although studies on these areas are still at the 17 Chapter Introduction laboratory/experimental level, the literature cited above, suggest a promising potential for the clinical applications of MSCs Due to the MSCs multi-differentiation potentials and their promising clinical usages, there will be a greater need for in vitro expanded MSCs, as the number of MSCs in vivo is not sufficient to support research and clinical usage Meeting this increasing need for more MSCs is one of the main motivations of the present study, in particular, in studying the role of S1P in adult stem cell proliferation Another major motivation is the pressing need for more knowledge and controls about MSCs multi-lineage differentiation in replenishing or replacing the damaged or dysfunctional organs In the next section, current strategies for human MSCs expansion and differentiation are addressed, including their advantages and their potential shortcomings 1.4.5 Current Strategies in MSCs Proliferation and Differentiation 1.4.5.1 Serum and Growth Factors for MSCs Proliferation The most widely used strategy for human MSC proliferation is using serum, which contains various growth factors that promote MSC proliferation Although currently there are many attempts to develop serum-free strategies, none of them seem to have achieved a similar proliferation level as serum However, serum composition is poorly defined, which means it contains unknown factors with unknown functions, and this may lead to potential side effect of serum on MSCs in culture Also, serum has a considerable degree of inter-batch variation, which makes it difficult for large-scale MSCs expansion in the same culture condition Moreover, most isolation and expansion protocols for clinically applicable MSCs utilize fetal calf serum as a supplement, which poses a potential risk of 18 Chapter Introduction infection as well as immunological reactions by different species Using human serum could overcome some of the potential problems caused by the use of animal serum; however, human serum is not easily obtainable in the quantities required for wide scale culture condition Moreover, there will still be a risk of inducing MSCs to unknown differentiation pathways due to serum’s poor definition problem, as well as a high risk of infection or pathogen transmission among different donors and recipients Due to these concerns, using serum to expand stem cells has never been without controversy Dimarakis et al (2006) and Sotiropoulou et al (2006) had a very interesting and hot debate on whether using animal or human serum could be acceptable for clinical application It is strongly believed that until all serum components are defined, or new potent stem cell expansion strategies are developed, these kinds of debate will not end From this point of view, although there are still no strategies that could expand MSCs as effectively as serum, it is still of value to develop a well-defined, serum-free or serumreduced medium for MSCs self-renewal Using exogenous growth factors or cytokines is a step forward in developing new strategies for stem cell expansion Various growth factors, such as PDGF (Lucarelli et al., 2003; Pébay et al., 2005; Kilian et al., 2004; Kang et al., 2005), fibroblast growth factor (FGF) (Van den Bos et al., 1997; Zaragosi et al., 2006; Tsutsumi et al., 2001), and transforming growth factor (TGF) (Gordon et al., 1997; Jian et al., 2006), have been shown to promote MSCs proliferation Although exogenous growth factors, or cytokines, have provided useful insights into developing serum-free MSC expansion strategies, they are too expensive for large-scale cells culture Moreover, their effects on stem cells are not fully known yet They might 19 Chapter Introduction exert effects on stem cells differentiation when used for stem cells proliferation For instance, the TGF-beta family, which has been shown to promote MSCs growth, also possesses the function to induce MSCs chondrogenic differentiation (Im et al., 2006) Similarly, the FGF family, which has been shown to be promising in promoting stem cell proliferation, but has also been implied, by numerous studies, to have an effect on stem cell chondrogenic and adipogenic differentiation (Chiou et al., 2006; Solchaga et al., 2005; Kakudo et al., 2007) Based on the above, using growth factors or cytokines as MSCs expansion strategies requires more knowledge about their potentials, especially with regard to stem cell differentiation Nevertheless, growth factors or cytokines provide attempts for expanding stem cells in a better-defined condition, which lowers the risk of stem cell transformation in undefined culture conditions, such as in serum However, the search still goes on in an attempt to develop more-defined, non-proteinaceous, cheaper strategies to promote stem cells proliferation 1.4.5.2 Exogenous Cytokines and Growth Factors, Non-proteinaceous Cocktails, Scaffold, Co-culture, or Genetic Modification for MSCs Differentiation Growth factors or cytokines, such as endothelial growth factor (EGF) and PDGF (Kratchmarova et al., 2005; Scavo et al., 2004), bone morphogenetic protein (Minamide et al., 2007; Dragoo et al., 2003) and FGF (Minamide et al., 2007), are usually used for MSCs differentiation However, these growth factors were shown not efficient enough to bias MSCs differentiation along aimed lineage, as some of the MSCs differentiation pathways are very similar in process For example, Mastrogiacomo et al (2001) and Shea et al (2003) found that many cytokines and growth factors which promote chondrogenic 20 Chapter Introduction differentiation were also somewhat implicated in osteogenic differentiation for MSCs, as the two processes are very similar In addition to protein-based cytokines and growth factors, some chemical compounds have also been shown to promote MSCs multipotent differentiation, such as 1,25dihydroxy vitamin D (Tsonis et al., 1991; Harmand et al., 1984), Prostaglandin E2 (Miyamoto et al., 2003; Biddulph et al., 2000), dexamethasone (Johnstone et al., 1998; Mackay et al., 1998), and ascorbic acid (Farquharson et al., 1998), in MSCs chondrogenic differentiation However, some of them, such as 1,25-dihydroxy vitamin D (Sammons et al., 2004), dexamethasone (Grigoriadis et al., 1988; Herbertson et al., 1995), and ascorbic acid (Herbertson et al., 1995) are also main components of cocktails for MSC osteogenic differentiation These studies supported the findings that certain differentiation lineages (such as chondrogenic and osteogenic differentiations) are quite close Therefore, strategies that may provide more control in stem cells differentiation are needed Another strategy to promote MSCs differentiation is to use a naturally occurring or artificially designed extracellular matrix (ECM) ECM mimics the natural in vivo environment and thus provides higher chances for a better cell attachment, growth, and differentiation Materials used to construct ECM could be either naturally occurring components (Latif et al., 2007; Kundu et al., 2006) or synthetic materials (Yim et al., 2007; Wang et al., 2006) Additionally, composite matrix scaffolds of both natural and synthetic materials could be fabricated to obtain an optimized condition for MSCs differentiation (Benoit et al., 2005; Mauck et al., 2007; Chastain et al., 2006) One of the 21 Chapter Introduction bio-safety concerns about using the synthetic components is their biocompatibility and biodegradability as they are foreign composites for the body In addition to the strategies stated above, the use of co-culture stem cells with a different cell population (Ong et al., 2006; Pisati et al., 2007; Li et al., 2007; Trivedi et al., 2007; Lee et al., 2007) is another practical method to induce MSCs differentiation along a certain lineage The main advantage of using co-culture systems is that it allows intimate contacts between different cells, found in an organ, providing an efficient way in creating a more physiological environment, thus facilitating differentiation to occur However, coculture systems have two main shortcomings due to the direct contact with culturing cells: high risk of pathogen transmission, and difficulty in separating the co-cultured cell populations Based on the co-culture strategy, using cell-conditioned media is a step forward (Hwang et al., 2007; Takagi et al., 2007; Nunes et al., 2007; Li et al., 2007), as it avoids intimate cell-to-cell contact, thus avoiding difficulties in separating co-cultured cells, but still retaining the advantages of fast transduction molecules transmission between culturing stem cells and the conditioned media Nevertheless, the risk of pathogen transmission, especially virus transmission, still exists when using filtered conditioned media A common limitation for the above strategies is the prolonged culture duration to get pure subpopulations, which may cause delayed treatment for patients, especially in autologous transplantation Moreover, prolonged culture duration could possibly alter the immunogenicity of the cells in culture and thus may result in immunerejection when the cells are transplanted back into the patient (Smythe et al., 2000) 22 Chapter Introduction Another alternative strategy for MSC differentiation, which overcomes this limitation of prolonged culture duration, is modulating the genome of the stem cells by using recombinant DNA constructs, encoding for the expression of certain proteins or growth factors that promote differentiation (Ramos et al., 2006; Gerstenfeld et al., 2001; Tsuchiya et al., 2003; Madry et al., 2002) However, genetic modulation has potential risks when using recombinant DNA technology in clinical applications Additionally, genetic modulation constantly up-regulates or down-regulates the expression of certain proteins or growth factors, which might induce unexpected malignant effects in the cells In summary, although current strategies for MSCs proliferation in a serum-free condition not seem comparable with the serum one, in terms of proliferation efficiency, attempts for developing well-defined, serum-free or serum-reduced strategies for MSCs proliferation are still valuable, since serum is obviously an obstacle for stem cells applications in the clinic As for current MSCs differentiation strategies, there is also much room for improvement in better-controlled strategies, which might bias MSCs to differentiate along certain lineage(s) to generate pure subpopulation(s) or shorten the duration needed for stem cells differentiating into a pure subpopulation Our strategy of utilizing non-protein-based compounds, namely the lysophopspholipid (S1P), and lysolipid-based SPHK inhibitors, would bring new insights to these two areas The objectives and significance of the study are addressed below 23 Chapter Introduction 1.5 OBJECTIVE AND SIGNIFICANCE The objective of the study was to shed light mainly on the following three aspects: 1) Develop and evaluate specific inhibitors of SPHK As was stated in sections 1.1 (SPHK and S1P) and 1.2 (SPHK and S1P regulation in pathological states), SPHK and its product S1P have been shown to be involved in various cellular events in physiological and pathophysiological processes Activation of SPHK and accumulation of S1P are believed to be involved in cell proliferation, including in embryonic stem cell proliferation (Pébay et al., 2005) In addition, inhibition of SPHK has been suggested to play a role in mediating embryonic stem cell differentiation (Pébay et al., 2005) To inhibit SPHK and S1P, the current strategies include using SPHK inhibitors to inhibit endogenous SPHK directly, or inhibiting the phosphorylated product S1P directly One of the successful attempts of inhibiting S1P is the development of the monoclonal antibody against S1P (Visentin et al., 2006), which had been introduced in Section 1.2.2 (SPHK and S1P in Cancer) It showed the monoclonal antibody to bind and neutralize extracellular S1P at its physiologically relevant concentrations, and helped to reduce tumor growth, invasion, and new vessel formation, in several murine models For SPHK inhibition, pharmacological studies have used limited numbers of compounds, including DMS, D, L-threo-dihydrosphingosine, and N,N,N-trimethylsphingosine However, these compounds are not specific inhibitors of SPHK as they are shown to affect protein kinase C (PKC) (Igarashi et al., 1989), sphingosine-dependent protein kinase (Megidish et al., 1995), 2-phosphoinositide-dependent kinase (King et al., 2000), and casein kinase II (McDonald et al., 1991) Moreover, these inhibitors are not capable 24 Chapter Introduction to distinguish between SPHK1 and SPHK2 Recently, a few natural product inhibitors developed from microbes (Kono et al., 2000a & b; 2001), have been developed as more specific SPHK inhibitors However, their biological activities and large-scale production capabilities are unknown yet Therefore, developing potent and specific inhibitors of SPHK that can be easily synthesized and produced in large scale would be highly desirable for basic research using as laboratory reagents, potential drugs for inflammation, some cancer diseases, as well as in stem cell research As the 3-D structure of SPHK remains unknown, it has been difficult to design targetspecific inhibitors, or structurally-based inhibitors In order to develop specific SPHK inhibitor, some researchers have gone on to screen chemical libraries of compounds (French et al., 2003); and other have designed analogues of the sphingoid backbone, the major common part for sphingolipids, as potential-selective SPHK inhibitors (Kim et al., 2005) In our study, a series of analogues of the natural substrate of SPHK- D-erythrosphingosine, were designed, synthesized, and evaluated as SPHK inhibitors They were tested for their inhibitory effects in vitro on cell extracts containing high levels of SPHK1 and SPHK2, as well as, on endogenous SPHK1 activity stimulated by the anaphylatoxin C5a, which is known to stimulate endogenous SPHK1 activity in neutrophils and macrophages (Melendez et al., 2004; Ibrahim et al., 2004) Moreover, the selected compounds were investigated on their inhibitory function on diacylglycerol kinase (DAGK), and human PKCα, as these two enzymes share a close-related catalytic domain with SPHKs, and some of the currently available SPHK inhibitors have been shown to inhibit DAGK and PKCα as well The selected compounds were tested for their 25 Chapter Introduction cytotoxicity as well In all investigations, the most commonly used SPHK inhibitor, DMS, was used as a positive control The compounds developed and evaluated as SPHK inhibitors in this study, could potentially be used as laboratory reagents for evaluating SPHK function in various cellular and pathological processes, or potential drugs for some diseases Moreover, in this thesis, their potential usage in the development of new strategies for facilitating stem cell differentiation is another research goal (next section) 2) Study the role of SPHK inhibitors in mediating human BM-MSCs and AD-MSCs differentiation One of the major risks using stem cells for therapy is their spontaneous differentiation ability to form teratoma, which are stem cell-derived tumors, especially for human embryonic stem cells (Wakitani et al., 2003; Vogel, 2005; Nussbaum et al., 2007; Hentze et al., 2007) The risk is inversely proportional to the differentiation stage, which indicates the more the differentiation commitment, the lower the risk of teratoma formation Therefore, mediating stem cells differentiation, in particular, promoting stem cells differentiation, in a controlled way, would be highly desirable in stem cells research and in their future clinical application As stated in section 1.4.5.2 (Exogenous Cytokines and Growth Factors, Non-pertinacious Cocktails, Scaffold, Co-culture, or Genetic Modification for MSCs Differentiation), current strategies for human MSC differentiation possess different limitations such as, prolonged culture duration, high cost, inability to consistently differentiate along specific pathways, risk of pathogen transmission and using recombinant DNA Therefore, it will 26 Chapter Introduction be beneficial to gain more knowledge on the molecule(s) that promote stem cells differentiation in order to develop new strategies for stem cell differentiation It has recently been shown that inhibition of SPHK stopped embryonic stem cell proliferation and induced the cells to undergo differentiation (Pébay et al., 2005) Thus, considering the laboratory interest in SPHK research and the above mentioned findings, my research project was derived and focused on studying the role of SPHK in adult stem cell differentiation, in an attempt to generate new information in the area of new strategies for stem cell differentiation A number of studies have reported a role for S1P in cell proliferation and differentiation, but not much has been reported for a role for SPHK in cell differentiation, especially in stem cells Very recently, Kuno et al (2007) reported that SPHK1 activation, by TGF-ß1, leads to Rho-associated myofibroblasts differentiation mediated by transactivated of S1PRs in the lung fibrogenic process To our knowledge, Pébay et al (2005) is the first to report that the direct inhibition of endogenous SPHK caused human embryonic stem cells differentiation However, it was obviously not their research goal to study how inhibition of SPHK worked in driving stem cells into differentiation, as there was no further research on it, except the only report that DMS stopped the human embryonic stem cells growth and caused them to lose their pluripotency The main goal in their study was to develop new strategies for stem cell proliferation, not for differentiation The hypothesis in this study is that when the rheostat of maintaining proliferation/differentiation in MSCs is altered by SPHK inhibitors, cell fate would be switched into cell differentiation or cell death Through mediating the concentration of SPHK inhibitor(s) used, cell death could be avoided The idea is that with the presence of 27 Chapter Introduction certain induction pressure by induction/differentiation factors, MSCs, treated with SPHK inhibitor(s), would differentiate into the designed subpopulation in a shorter period Thus, the main objective of the study in this part was to investigate whether the inhibition of endogenous SPHK activity, affected human MSCs differentiation; if so, how much they affected the differentiation This study might then provide a new strategy, at a reasonable cost, to promote the stem cells differentiation by shortening the duration needed for the stem cells differentiating into its pure subpopulations To achieve the objective stated above, the inhibition of SPHK was studied in human BMMSC and AD-MSC osteogenic and adipogenic differentiation pathways, as these two pathways are two typical ones which human MSCs could differentiate into A wellestablished cocktail that contains 0.5mM isobutyl-methylxanthine, 1μM dexamethasone, 10μM insulin, and 200μM indomethacin was used to induce both human BM-MSCs and AD-MSCs to differentiate along adipogenic pathway (Kim et al., 2007; Kakudo et al., 2007) Another well-established cocktail that contains 0.01μM 1,25-dihydroxyvitamin D3, 50μM ascorbate-2-phosphate, and 10mM ß-glycerophosphate was used to induce the stem cells to differentiate along osteogenic pathway (Heng et al., 2004; Cao et al., 2005) Cells differentiation along either adipogenic or osteogenic pathway, with/without the presence of SPHK inhibitors/cocktail was compared, to evaluate the role of the SPHK inhibitors in human MSCs adipogenic and osteogenic differentiation The originality of my research project is that it is designed to investigate in detail the role of SPHK in promoting human BM-MSCs and AD-MSCs proliferation and differentiation It would be promising and meaningful for clinical usage, as inhibiting SPHK might at the 28 Chapter Introduction same time suppress up-regulated immune responses, which usually happens in stem cell transplantation Also, it should be noted, that human AD-MSCs are a new source of human MSCs (Zuk et al., 2001) My study on their differentiation would definitely provide more knowledge of this new source of human MSCs 3) Study the role of S1P in human BM-MSCs and AD-MSCs proliferation Human stem cells are increasingly becoming the focus of research for their use in developmental biology, and in clinical settings for regenerative medicine However, very little is known about the factors that stimulate their growth and maintain of their multipotency, which limits the development of the strategies for stem cell expansion in better controlled, serum-free, conditions In this study, the role of S1P in human BM-MSCs and AD-MSCs proliferation was studied S1P alone, S1P with a lower percentage of fetal bovine albumin, and S1P with other growth factor like PDGF-AB were compared as strategies for human BM-MSCs and AD-MSCs proliferation It might be possible that S1P only could not promote a significant cell proliferation rate in human BM-MSCs and AD-MSCs, as it did not promote human embryonic stem cell growth significantly (Pébay et al., 2005) However, human BM-MSCs and AD-MSCs, as tissue-specific stem cells, are very different from human embryonic stem cells in terms of cell differentiation properties and in vitro culture conditions Human BM-MSCs and ADMSCs grow and attach to the culture flask as single layer cells, whereas embryonic stem 29 Chapter Introduction cells are cultured on the feeder layer cells, and form embryoid body which contains a group of cells In this study, the hypothesis is that, S1P may work with serum or other growth factors synergistically or additively, to promote MSC proliferation, as S1P could crosstalk with other growth factors like vascular endothelial growth factor (VEGF) (Spiegel and Milstien, 2003) to increase cell growth, and regulate cell movement In this study, PDGFAB was chosen to work with S1P to study the effect of them on stem cell proliferation, as it had been shown to promote stem cell proliferation (Kang et al., 2005; Pébay et al., 2005) The major advantage of using S1P in MSCs expansion is that S1P is a lysolipid, easily purified or synthesized, can be purchased from most chemical companies, and is much cheaper than growth factors Thus using S1P in MSCs expansion would significantly reduce the cost, relative to that in most currently used defined strategies, which mostly use a combination of several growth factors It should be noted that only PDGF-AB, combined with S1P, was chosen to be investigated in the study There might be other growth factors that may work well with S1P to promote MSC proliferation as well, but they are not within the current research scope In summary, this study aimed to develop and evaluate some novel and more specific inhibitors for human SPHK for research use, to compensate the current lack of specific inhibitors of SPHK commercially available The synthetic inhibitors could be used in the future for laboratory reagents, potentially in the development of novel therapeutics, and 30 Chapter Introduction last but not least in mediating stem cells differentiation Moreover, this study investigated the role of S1P in human BM-MSCs and AD-MSCs proliferation, with the aim to develop a better defined, serum-free and low growth factors, and cheaper strategy for promoting stem cell proliferation The study program was divided mainly into three parts: 1) The development and evaluation of human SPHK inhibitors, which will be addressed in chapter 2) The role of SPHK inhibitors in human BM- and AD- MSC differentiation, which will be addressed in chapter 3) The role of S1P in human BM- and AD- MSC proliferation, which will be addressed in chapter The conclusion for the present study and possible future work will be addressed in chapter 31 ... clinical usage Meeting this increasing need for more MSCs is one of the main motivations of the present study, in particular, in studying the role of S1P in adult stem cell proliferation Another... maintenance and differentiation In particular, we are interested in the role of S1P in human adult stem cell proliferation and stemness maintenance, and how SPHK inhibition would affect human adult. .. considering the laboratory interest in SPHK research and the above mentioned findings, my research project was derived and focused on studying the role of SPHK in adult stem cell differentiation, in