Tsc1 Is A Critical Regulator Of The Survival And Function Of Macrophage

Objective:To explore the role of retinoic acid (RA) and SB431542 in the differentiation of mesenchymal stem cell (MSC) like cells from mouse embryonic stem cells (ES) and induced pluripotent stem cells (iPS)Methods:The ES cells and iPS cells were differentiated in medium with 10 uM SB431542 or with RA at different concentrations which rang from 0.05 nM to 0.40 nM. Replace the medium once a day. After 4 days of differentiation, the expression of the CD105 and Sca-1 on the cells were analyzed by flow cytometry. CD105+ cells and Sca-1+ cells were considered as mesenchymal stem cell like cells (MSC-like cells).Results:1) ES and iPS were differentiated into epithelial-like cells in the control medium and in the SB431542-supplemented medium. ES and iPS were efficiently differentiated into MSC-like cells in the RA-supplemented medium;2) After 4 days of ES differentiation, the percentage of Sca-1+ cells in the SB431542-supplemented medium was increased significantly compared with that in the control medium (P<0.05), and the percentages of CD105+ cells in the SB431542-supplemented medium showed no significantly difference compared with that in the control medium(P>0.05).The percentages of CD105+cells in the medium containing 0.05,0.10,0.20 or 0.40 nM RA were all increased significantly compared with that in the control medium and that in the SB431542-supplemented medium (P <0.05).The percentages of Sca-1+ cells in the medium containing 0.05,0.10,0.20 or 0.40 nM RA were all increased significantly compared with that in the control medium (P<0.05), and were all significantly decreased compared with that in the SB431542-supplemented medium (P<0.05). The percentages of CD105+ cells and Sea-1+ cells in the medium containing 0.20 nM RA were all increased significantly compared with those in the medium containing 0.10,0.20 or 0.40 nM RA (P<0.05);3) After 4 days of iPS differentiation, the percentages of Sea-1+ cells and CD 105+ cells in the SB431542-supplemented medium was increased significantly compared with those in the control medium (P<0.05). The percentages of CD105+ cells in the medium containing 0.05,0.10,0.20 or 0.40 nM RA were all increased significantly compared with that in the control medium and that in the SB431542-supplemented medium (P<0.05). The percentages of Sca-1+ cells in the medium containing 0.05, 0.10,0.20 or 0.40 nM RA were all increased significantly compared with that in the control medium (P<0.05), and were all significantly decreased compared with that in the SB431542-supplemented medium (P< 0.05); The percentage of Sea-1+ cells in the medium containing 0.20 nM RA was increased significantly compared with those in the medium containing 0.10,0.20 or 0.40 nM RA (P<0.05). The percentage of CD105+ cells was increased significantly compared with that in the medium containing 0.10 or 0.20 nM RA (P<0.05) and with no significantly difference with that in the medium containing 0.40 nM RA (P>0.05)Conclusion:RA promotes the differentiation of ES and iPS into MSC-like cells, which provides a practical means for the study of stem cell differentiation.Background/Aims:Tuberous sclerosis complex 1 (Tsc1) and Tuberous sclerosis complex 2 (Tsc2) form a complex to regulate metabolism and energy homeostasis via inhibition of mammalian target of rapamycin complex 1 (mTORC1). Loss-of-function mutations in TSC1 or TSC2 result in elevated activity of mTORC1, which leads to increased cell growth and proliferation associated with the development of numerous noncancerous tumors in tuberous sclerosis patients. Tsc1 has been shown to regulate M1/M2 polarization of macrophages, but the precise roles of Tscl in the function and stability of macrophage are not fully understood. Here we show that Tscl is required for regulating the survival, migration and phagocytosis of macrophages.Methods:Mice with Tsc1 homozygous deletion in myeloid cells (LysMCreTsc1flox/flox; Tsc1 KO) were obtained by crossing Tsc1flox/flox mice with mice expressing Cre recombinase under the control of Lysozyme promoter (LysMCre). The WT and Tscl KO mice were determined using PCR. Deletion of Tscl by LysMCre-mediated recombination in macrophages was confirmed by western blot assays. The apoptosis and growth of macrophages were determined by flow cytometry and RT-PCR. Arginase activity of primary macrophages was determined by quantitative colorimetric assay at 430 nm using a QuantichromTM arginase assay kit (Bioassay Systems) according to the manufacturer’s instructions. The phagocytosis of macrophages was determined using a VybrantTM phagocytosis assay kit. The migration of macrophages was determined using transwell migration assay.Results:Peritoneal macrophages of Tsc1 KO mice exhibited increased apoptosis and enlarged cell size. Both Ml and M2 phenotypes in Tscl-deficient macrophages were elevated in steady-state as well as in inflammatory conditions. Tscl-deficient macrophages demonstrated impaired migration and reduced expression of chemokine receptors. Furthermore, phagocytosis activity and reactive oxygen species (ROS) production were enhanced in Tsc1-deficient macrophages. The activation and proliferation of CD4+ T cells cocultured with Tsc1-deficient macrophages were impaired. Furthermore, pharmacological inhibition of mTORC1 partially reversed the aberrance of Tsc1-deficient macrophages.Conclusion:1) Tscl promotes survival and inhibits apoptosis of macrophage. The roles of Tsc1 in regulating survival and inhibiting apoptosis of macrophage are dependent on mTORC1. CD71 and CD98 may play important roles in Tsc1 regulating the growth of macrophage.2) Tsc1 regulates the polarization of macrophage. Tsc1-deficient macrophages displayed elevation of both M1 and M2 phenotypes in steady-state conditions and in inflammatory conditions.3) Tsc1 promotes the migration of macrophage.Tsc1-deficient macrophages demonstrated reduced expression of chemokine receptors and impaired migration induced by CCL2. The expression of chemokines of Tsc1-deficient macrophages was increased. Furthermore, phagocytosis activity and ROS production were enhanced in Tscl-deficient macrophages. Tsc1 in macrophage promotes the activation of CD4+ T cell.4) Myeloid expression of Tsc1 is required to prevent lymphoproliferative disorder. The spleens, LNs were larger in Tsc1 KO mice than those in WT mice. More activated CD4+ T cells and CD8+ T cells in the spleens and LNs of Tsc1 KO mice than those in WT mice. However,5-12 weeks old Tsc1 KO mice showed no survival deficiencies and no loss of weight compared with WT mice.