Development of sphingosine kinase (SPHK) inhibitors and the role of sphingolipids in adult stem cell proliferation and differentiation 5

Chapter Conclusion and Future Work CHAPTER CONCLUSION AND FUTURE WORK The aim of this project was to develop new inhibitors for SPHK and investigate the roles of SPHK and its product S1P in adult stem cell proliferation and differentiation. Each of my result chapters contain separate conclusion sections, this chapter summarizes all of the results presented in this thesis and discuss the potential application and future work derived from my research project. In the first part of the study (Chapter 2), a series of SPHK inhibitors were designed and six of them (CP1, CP2, CP3, CP4, CP5 and CP6) were synthesized and evaluated for further testing. I found that all of the six compounds are specific inhibitors of SPHK1, and did not inhibit SPHK2, DAGK, or PKC alpha (the latter two enzymes were chosen as counter-screens for compound specificity). Among the compound studied, CP3 and CP6 showed the best inhibitory effect on the endogenous SPHK1, activated by the signal transduction cascade, triggered by the anaphylotoxin C5a in human macrophages. The compounds and the data generated from this part of my project form the basis for an International Patent Application submitted in August 2007 (US Provisional Patent Application Title: Synthesis of Sphingosine Analogues Inhibitors of Sphingosine Kinases and Their Therapeutic Usages; Application No.: US 60/959,115). In the second part of my project (Chapter 3), the generated SPHK inhibitor CP6 was studied for its functions in human BM- and AD- MSCs osteogenic differentiation and adipogenic differentiation. CP6 was selected due to its highest synthesis-yield and for 153 Chapter Conclusion and Future Work being one of the most potent and specific SPHK1 inhibitors (chapter 2), DMS was continuously used as a reference inhibitor. It is interesting to note that CP6 and DMS affected the osteogenic differentiation and adipogenic differentiation in human MSCs differently. In human BM- and AD- MSCs osteogenic differentiation, 0.5μM CP6 and DMS showed to promote the osteogenic differentiation in most of the cases, while at higher amounts (1μM DMS and 5μM CP6) showed to inhibit the osteogenic differentiation in most of the experiments. Interestingly, in human BM- and AD- MSCs adipogenic differentiation, DMS and CP6 showed to promote the adipogenic differentiation in both types of MSCs in a dose-dependent manner. The reason for why at high concentrations CP6 and DMS inhibited the stem cells osteogenic differentiation remains unclear. In the future, it would be interesting to study how lower concentrations of CP6 (≤0.5μM), or any other of the compounds generated in this study, affect human MSCs differentiation in the mixture of both osteogenic differentiation media and adipogenic differentiation media, which may simulate a more in vivo environment. By optimizing the concentrations of SPHK inhibitor(s) used, we could possible develop a new strategy controlling human MSCs osteogenic and adipogenic differentiation switching. To our knowledge, this is the first report on inhibitors of SPHK mediating human BMand AD- MSCs differentiation into osteogenic and adipogenic paths. Thus, the results presented here contribute to the current understanding of human MSCs osteogenic/adipogenic differentiation. The data described in chapter might also pave the way, for a more controlled and facilitate differentiation of stem cells, for future 154 Chapter Conclusion and Future Work therapeutic applications to balance the abnormal portions of osteoblasts and adipocytes observed in bone diseases such as osteoporosis and osteopenia, where the bone volume is decreased but the adipose tissue increases (Jaiswal et al., 2000). It also suggests the potential for the use of SPHK inhibitors in preconditioning stem cells before transplanting them into the body, by committing stem cells along specific differentiation path. More work, including in vivo work, needs to be done to fully validate the potential for SPHK inhibitors in adult-stem cell differentiation. In the final part of my research project (Chapter 4), the bioactive lipid S1P was studied for its role in promoting human BM- and AD- MSCs proliferation. The results presented in chapter show that in serum-free growth condition, S1P alone may be able to induce only limited level of proliferation (1.2-1.3 fold growth as compared with the serum-free media). However, S1P could work synergistically with “normal” serum containing media (10% FBS), but more importantly, S1P showed to be able to induce an excellent level of cell proliferation in serum-reduced media (1% FBS), especially in the human AD-MSCs, where 1μM S1P and 1% FBS could promote around 50% more cells growth (Figure 4.6B) when compared to the cells cultured in 1% FBS. S1P could also work additively with PDGF-AB to promote an even higher level of cell proliferation. Another interesting observation, described in chapter 4, is that human MSCs cultured in S1P-supplied media managed to restore the morphology changes observed when the cells were cultured in serum-free media. Moreover, S1P did not show to change the cell surface markers expression profile in human BM- or AD-MSCs. Furthermore, 1μM S1P 155 Chapter Conclusion and Future Work restored the osteogenic differentiation potential lost in human AD-MSCs cultured in 1% FBS (Figure 4.11B). The results presented in chapter suggest that S1P alone might not be potent enough for human BM- and AD- MSCs proliferation, however, when used in combination with serum containing media, 10% FBS or 1% FBS, or with the growth factor PDGF-AB, S1P promoted a much better level proliferation than using serum or PDGF. Although, in this study, S1P was not shown to be as potent in promoting adult-stem cell proliferation, as it was shown for embryonic stem cells (Pébay et al. 2005), it is shown in this study that S1P in combination with PDGF-AB, or reduced serum, can be an important element in the development of better defined serum-free or serum-reduced strategies to promote human-adult BM- and AD- MSCs proliferation. S1P is much cheaper than most growth factors; therefore including S1P in stem cells culture media would lower the cost of MSC proliferation. Moreover, the functions of S1P are quite well understood compared with many growth factors. This would reduce the risk of inducing unknown functions in MSC expansion, when using some growth factors with less known functions. More work need to be done to optimize a combination strategy of using S1P with other growth factors, to promote human MSCs proliferation. Another contribution of this study is that it provides more information on human ADMSCs, which is a new source of MSCs recently established (Zuk et al. 2002), with very little studies on their expansion being reported in the literature. The benefit of using this type of MSC is quite obvious, as the cells are extracted from “unwanted” fat. Also, the 156 Chapter Conclusion and Future Work procedure of extracting these cells is much less painful, compared with extracting MSCs from BM. This study investigated the function of S1P, S1P+10%FBS, S1P+1%FBS and PDGF-AB in human AD-MSCs and the results suggest a potential for S1P in stem cell proliferation culture conditions. These findings would potentially help speed up the research on human adipose-derived MSCs by inducing fast proliferation with a defined serum-free or serum reduced strategy. Finally, although this study was mainly done on tissue-specific stem cells (i.e. MSCs), the knowledge accumulated on cell expansion and differentiation might also be applicable in other types of stem cells. Also, it should be noticed that both human BMand AD- MSCs are primary cells, and thus they may possess certain variations between different donor cells. Therefore, more work need to be done to further confirm our conclusions. Finally, the study could be expanded to study other stem cells and other differentiations pathways, to further validate and confirm the potential usage of SPHK inhibitors in stem cell differentiation, and S1P roles on stem cell proliferation. 157 . Title: Synthesis of Sphingosine Analogues Inhibitors of Sphingosine Kinases and Their Therapeutic Usages; Application No.: US 60/ 959 ,1 15) . In the second part of my project (Chapter 3), the generated. Finally, the study could be expanded to study other stem cells and other differentiations pathways, to further validate and confirm the potential usage of SPHK inhibitors in stem cell differentiation, . CP6 and DMS inhibited the stem cells osteogenic differentiation remains unclear. In the future, it would be interesting to study how lower concentrations of CP6 (≤0 .5 M), or any other of the