| Part I the kinetics of mTORC1 activity in hematopoitetic stem cells during ex vivo expansionObjective:mTORCl intrgrated many cellular signals. Previous studies have have shown that hyperactivity could break the cells homeostasis. The stemness of hematopoietic stem cells gragually lost during ex vivo expansion. In this study, our target is to investigate the kenetics of mTORC1 activity in hematopoitetic stem cells during ex vivo expansion.Methods:Separated the Lin-Sca-1+ cells from mouse bone marrow. Put these cells in serum free medium supplement with the cocktail cytokines, and cultured for ten days, at the 6th day, Rapamycin was added, DMSO was also added as a control.10 days later, collected all of progeny cells and detected the phosphorylation of p70 s6k expression in hematopoietic stem cells.Results:Lin-Sca-1+ cells proliferated robustly under the stimuli of cytokines, at 6th day, the fold of cells increase was 25±4.5, and at 10th day, the fold of cells increase was 82±5.2. the expression of p-p70 s6k was paralleled with it. under the unexpanded conditions, only small part of cells expressed p-p70 s6k, about 5.82±3.12%, and at 6th day,22.4±4.67% cells expressed p-p70 s6k, notably, at 10th day,73.4±5.23% cells expressed p-p70 s6k. The addition of Rapamycin at 6th day significantly decreased the activity of mTORCl, but did not abrogated it.Conclusion:mTORCl activity was moderately increased during the early phase of expansion, but greatly increased during the late pahse of expansion, parrelled with the robust expansion of total cells. PartⅡInhibiting mTORC1 activity at the late phase of expansion improved hematopoietic stem cells ex vivo expansionObjective:the hyperactivity of mTORC1 would lead hematopoietic stem cells exhaustion. Rapamycin can significantly decreased the activation of mTORC1. in this part of study, Rapamycin was added at the late phase of expansion, to see whether this inhibition could improve the results of ex vivo expansion.Methods:Separated the Lin-Sca-1+ cells from mouse bone marrow. Put these cells in serum free medium supplement with the cocktail cytokines, and cultured for ten days, at the 6th day, Rapamycin was added, DMSO was also added as a control.10 days later, collected all of progeny cells and detected the expansion efficiency and the function of progeny cells in vitro and in vivo.Results:Rapamycin decreased the total nucleated cells slightly compared to Veh, the total nucleated cells in Rapamycin group was 2.43±0.21×105, while in Veh group the number was 2.73±0.33×105. but Rapamycin increased the percentage of Lin-Sca-1+cells and LSK+cells. When cauculated the number of Lin-Sca-1+cells and LSK+cells, it was found that Rapamycin acquired 1.97±0.56×104 Lin-Sca-1+cells and 10.64±3.42×103 LSK+cells, Veh acquired 1.27±0.47×104 Lin-Sca-1+cells和7.34±3.0×103 LSK+cells. Comparing to the input cells, Veh had got 4.2 fold of expansion and Rapamycin had got 6.5 fold of expansion. In CAFCs assay, Rapamycin also acquired more day14 CAFCs and day35 CAFCs, about 150.67±16.4 and 9.26±1.45 per 100,000 progeny cells respectively, Veh just acquired 97.3±7.6 and 3.53±1.02 per 100,000 progeny cells respectively. Comparing to the input cells, Veh only had got 0.73±0.08 fold of day35 CAFCs expansion, but Rapamycin had got 1.95±0.25 fold of day35 CAFCs expansion. The engraftment capacity was significantly improved by Rapamycin refleted by the fact that more donor cells was detected in the mouse that injected the Rapamycin cultured cells.at 8th weeks and 32th weeks, Rapamycin cultured cells acquired 32.23±10.7% and 42.1±11.2% respectively;but Veh cultured cells only acquired 18.16±9.32% and 9.93±8.25% respectively.Conclusion:Rapamycin improved the ex vivo expansion of hematopoietic stem cells and maintained the stemness.PartⅢthe mechanism of improving ex vivo hematopoietic stem cells expansionObjective:mTORCl hyperactivity was correlated to stem cells apoptosis, differentiation and senescence under other celluar context. In this study, we investigated whether improving ex vivo hematopoietic stem cells expansion involved these mechanisms.Methods:Separated the Lin-Sca-1+ cells from mouse bone marrow. Put these cells in serum free medium supplement with the cocktail cytokines, and cultured for ten days, at the 6th day, Rapamycin was added, DMSO was also added as a control.10 days later, collected all of progeny cells and detected the related molecules expression.Results:total cells and LSK+cells apoptosis were detected by Annexin-V; the results showed that Veh led 8.93±1.5% total cells and 2.61±0.57% LSK+cells to apoptosis, and rapamcyin led 11.79±3.7% total cells and 2±0.22% LSK+cells to apoptosis. In CFCs assay, it showed that Rapamycin had no effect on the formation of CFU-GM and BFU. In SA-β-gal staining assay, we have not detected positive cells in unexpanded cells,but after 10 days expansion, it showed 9.7±1.95% Lin-Sca-1+cells presented SA-β-gal positive. In immunofluorescence assay, it showed that the LC3b expression, a widely used marker for autophagy detection, decreased in Rapamycin group. In cell cycle detection, it showed that Rapamycin increased the percentage of Go phase cells, about 21.7±3.5%, but in Veh group the percentage was only 11.75±3.3%. addtionally, Rapamycin also decreased the expression of p16, p53, cyclinC and cyclin D1.Conclusion:inhibition of mTORCl improved ex vivo hematopoietic stem cells expansion did not through suppression of apoptosis and differentiation but through suppression of senescence.Part IV the crosstalk between mTORCl and p38 MAPK a during ex vivo hematopoietic stem cells expansionObjective:In our previous studies, we have found that p38 MAPKαactivation was responsible for the senescence of hematopoietic stem cells during ex vivo expansion. Here we have proved that mTORCl was also involved in senescence regulation. So in this part of study we investigated the crosstalk between mTORCl and p38 MAPK a.Methods:Separated the Lin-Sca-1+cells from mouse bone marrow. Put these cells in serum free medium supplement with the cocktail cytokines, and cultured for ten days, at the 6th day, Rapamycin was added, DMSO was also added as a control. For SB203580(SB) treatment, SB was added at the beginning of culture. 10 days later, collected all of progeny cells and detected the related molecules expression.Results:in western blot and immunofluorescence assay, it was found that Rapamycin did not affected the expression of p-p38 MAPKα, but SB further increased phosphorylation of p70 s6k. co-inhibition of mTORCl and p38 MAPKαcould further increased ex vivo hematopoietic stem cells expansion. In Veh, Rapamycin, SB and combination group, it had got 1.5±0.3×103,1.9±0.1×103,2.2±0.2×103 and 2.6±0.14×103 LSK+ cells respectively. For day35 CAFCs, it got 3.4±0.57,7.8±0.32,9.7±0.21 and 11.4±0.63 per 100, OOOcells respectively.In SA-β-gal staining assay, combinated treatment further decreased the SA-β-gal positive cells, to 1.9±0.7%. in real time PCR assay, combinated treatment also further decreased the expression of pl6, p53 than either of them.Conclusion:inhibition m TORC1 did not affect the activity of p38 MAPKα, but inhibition of p38 MAPK a further increased the activity of m T0RC1. Coinhibition could get more greater extent of hematopoietic stem cells expansion through enhanced suppression of senescence.
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