The Preliminary Study Of Proliferation In Vitro Of Hematopoietic Stem Cells Under Co-processing Of Small Molecule Inhibitors

Hematopoietic stem cell transplantation is a more effective method for treating blood disorders in clinical,stem cell transplantation use the ability of hematopoietic stem cells(HSCs)to multi-hematopoietic reconstruction.In the adult,hematopoietic stem cells found primarily in the bone marrow,however,a limited number available in the bone marrow hematopoietic stem cells,can not solve the clinical demand,This requires us to mimic the microenvironment of the bone marrow in vitro without affecting hematopoietic stem cell function,to solve the clinical needs.However,the bone marrow microenvironment is a very large complex and coordinated regulatory network.In the bone marrow microenvironment various proteins,molecules,signaling pathways cooperative regulation of hematopoietic stem cell homeostasis.in the body severe blood loss,hematopoietic stem cells can proliferate rapidly differentiate into various blood cells,in the body steady,hematopoietic stem cells can stable stationary state.Studies have reported,P38 MAPK,mTOR,GSK3β,HDAC signaling pathways in hematopoietic stem cell has negative regulatory role,they can promote the differentiation of hematopoietic stem cells,inhibit the proliferation of hematopoietic stem cells,inhibit the function of hematopoietic stem cells,reduce the ability to reconstruct the hematopoietic system.When a signaling pathway suppressed by signal pathway inhibitor,We found it can proliferate to promote hematopoietic stem cells,and enhance the ability of reconstitution the hematopoietic system,however the effective number of hematopoietic stem cells only increased 1fold.It still can not solve the clinical demand.Hematopoietic stem cells under the signal coregulate network in the bone marrow microenvironment,we think that more than one signaling pathway may be more efficient to promote efficient amplification of hematopoietic stem cells.First of all,we have optimized the culture conditions and the culture method of hematopoietic stem cells in order to adapt to the culture environment of our laboratory,we sorted 1000 LSK cells cultured for 9 days,then analysis at day 10,cells were found in a good condition.Secondly,we buy P38 MAPK,mTOR,GSK3β,HDAC signaling pathways inhibitors and through experiments to find more appropriate inhibitory concentration.At last,We researched the effect to hematopoietic stem cells under different combinations of inhibitors,include in vitro flow analysis,competitive transplants and clonogenic assay.Through experimental study we found co-inhibit signal pathways can efficient promote proliferation of hematopoietic stem cells,especially co-inhibit GSK3βand P38 signal pathway inhibitors may be effective amplification 2-3 folds,it can enhance the rate of chimeric and the ability of reconstruction of the hematopoietic system.When we choose the inhibitory concentration of HDAC signal pathway inhibitor,we are excessive dependence on cell state thereby result in a low chimeric.Hematopoietic stem cells in combination with other signaling pathways inhibitor without HDAC inhibitor treatment also have a corresponding amplification,but they have low chimeric,I believe that after appropriate adjustments the concentration of the inhibitor can improve the amplification efficiency of hematopoietic stem cells.