| Hematopoietic stem/progenitor cell(HSPC)transplantation has been considered as an ideal option for certain disorders treatment including malignant and hematologic immunodeficiency diseases.Cord blood(CB)has several unique advantages such as high HSPC content,high proliferative potency,the ease of collection,and minimal risk of infection transmission to the recipient,making it as an excellent alternative source of HSPC transplantation.However,low absolute CD34+ cell numbers per CB graft collection pose a distinct disadvantage,especially for larger sized adult transplant recipients.A crucial approach to break through the bottleneck is to expand CB HSPC ex vivo prior to infusion.The current studies applied cytokine cocktails such as stem cell factor(SCF),thrombopoietin(TPO),and FMS-like tyrosine kinase-3 ligand(FL)in culture system to expand HSPC.These cytokines activate the interrelated intracellular signaling pathways by binding to surface receptors,leading the entrance of cell cycle for expansion.SCF and FLT-3 activates phosphatidylinositol 3-kinase(PI3K)to induce HSPC expansion.Glycogen synthase kinase-3(GSK-3)is a well-established downstream component of the PI3K signaling pathway but is also a key enzyme that negatively regulates the canonical Wnt signaling pathway.Wnt signaling is thought to be activated through PI3K signaling-mediated GSK-3 inhibition.In addition,TPO can also mediate the activation of MEK/ERK and NF-κB pathways.The rapid differentiation and loss of reconstitute potential of HSPC is inevitable in ex vivo culture conditions.The role of these signaling pathways in HSPC expansion and function maintenance and how to enabled a precise balance in the fate decision need to be investigated.In this thesis cord blood derived CD34+cells were expanded in serum-free culture system with SCF/TPO/FLT-3 cytokines combination and small molecule signaling regulator to study effect of different dosages of Wnt,MEK/ERK,and NF-κB signaling pathways on HSPC expansion and biological function.Firstly,the effects of Wnt pathway agonist,BIO,on the growth factor-driven expansion and function of HSPC were determined.5～100 nM BIO was added to the culture at day 0 and day 4.and the result showed 100 nM BIO could regulate expansion of CD34+cells.100 nM BIO in cultures exhibited a significantly increased contribution to the expansion of total cells and CD34+ cells on day 4.However,BIO treatment for 7 days attenuated total cells and CD34+cells expansion,while enhancing the multilineage commit potential and secondary expansion ability of expanded CD34+cells.The expression of CTNNB,AXIN2,APC and GSK3,which are Wnt signaling factors related to β-catenin,were differentially expressed in BIO expanded CD34+ cells when compared the control.It was found that β-catenin levels underwent a notable increase in cultured cell compared to that of fresh CD34+cells.It is interesting that continuous BIO treatment led to a marked Wnt activation and accumulation ofβ-catenin,with reduced CD34+cell expansion and better functional maintenance.Based on this finding,an optimized feeding strategy that BIO was added to the culture on days 0 and 7 was proposed to support substantial expansion of HSPCs.The expansion of CD34+cells,CD34+CD38-cells and CD34+CD38-CD45RA-CD90+CD49f+ cells in the optimized BIO feeding strategy on day 10 were 28.7±0.4-folds,23.8 ± 0.8-folds,and 4.5±1.0-folds,which was significantly higher than control(16.2 ± 0.7-folds,13.4 ± 0.8-folds,and 2.2±0.2-folds)and BIO was added to the culture on days 0,4,and 7(11.8±1.7-folds,10.6±1.6-folds,and 1.9±0.4-folds).Moreover,the optimized BIO feeding strategy achieved increased HSPC expansion without the loss of biological functions.The content of β-catenin in CD34+cells was at a moderate level in the optimized BIO feeding strategy,and the expression of expansion related genes downstream of Wnt signaling pathway was up-regulated,while cell apoptosis was significantly reduced,suggesting that moderately activation of Wnt signaling pathway promote HSPC ex vivo expansion.Further,we used the pharmaceutical and genetical intervention targeting MEK1 to regulate MEK1 expression and the kinase activity.The role and underlying regulatory mechanism of MEK/ERK signaling pathway in HSPC ex vivo expansion was investigated.Both treatment with inhibitor of MEK1,Trametinib,and transfection with small interfering RNA(siRNA)led to a drastic decrease in the CD34+cells numbers,implying that MEK1 inhibition prevented the ex vivo expansion of HSPC.We treated HSPC with a small molecular activator,PAF-16,which is capable of MEK activation.PAF C-16 regulated the proliferation of HSPC in a dose-dependent fashion.500 nM PAF C-16 treatment generated 2.22-folds,2.53-folds,and 2.04-folds greater numbers of CD34+cells,CD34+CD38-cells and CD34+CD38-CD45RA-CD90+CD49f+ cells,respectively,compared to cytokine only conditions on day 10.Results showed that PAF C-16 expanded cells had similar percentage of phenotypical HSPC and expansion folds in secondary ex vivo cultures compared with control.Numbers of erythroid burst-forming unit(BFU-E)in CFU assay and the percentage of CD71+CD235a-primitive erythroid progenitor cells in total population after 10-days were significantly increased,demonstrating that CD34+cells cultured with PAF C-16 improved the erythroid differentiation potency of HSPC.Furthermore,the expression and content of c-Myc in CD34+ cells,the proportion of CD34+cells entering the cell cycle,and cell proliferation rate increased,suggesting that activation of MEK/ERK signaling pathway can promote the ex vivo proliferation of HSPC through up-regulation of c-Myc.In addition,NF-κB signaling specific small molecular inhibitor TPCA-1,which targeting IKKβ,was used in culture and the effect of NF-κB inhibition on CB-HSPC metabolism and expansion was investigated.50,100,300,500 and 1000 nM TPCA-1 were added to the culture system at day 0,4 and 7.After 10 days of culture with 100 nM TPCA-1,the expansion of CD34+CD38-cells and CD34+CD38-CD45RA-CD90+CD49f+ cells were significantly increased compared to the cytokine priming alone.Notably,TPCA-1 treatment generated～2-fold greater percentage of CD34+CD38-cells,CD34+CD38-CD45RA-CD90+CD49f+ cells,and CD34+EPCR+compared to cytokine only condition.Moreover,NF-κB inhibition increased the expression of self-renewal and function maintenance related genes PU.1、Hoxa9、MEIS1、HOXB4.TPCA-1 expanded CD34+cells displayed increased serial colonies forming potency and secondary expansion capability.In order to investigate whether NF-κB inhibition in culture leads to metabolic reprogramming,the mitochondrial properties and metabolic flux were determined.It was found that the expression levels of the key regulator of mitochondrial biogenesis(Pgc1α)and mitochondrial chaperones and proteases(ClpP,Hsp10,Hsp60)were decreased in TPCA-1 expanded CD34+cells.Mitochondrial mass and membrane potential were markedly decreased with TPCA-1 treatment,leading to the reduced mitochondrial reactive oxygen species(ROS)level in HSPC.NF-κB inhibition led to augmented glycolysis rate with compromising mitochondrial metabolism in CD34+cells,suggesting that NF-κB pathway inhibition improved glycolysis and limited ROS production to promote the ex vivo expansion and maintenance of functional HSPC.In summary,this study investigated the effects of ex vivo regulation of cytokine induced signaling pathways on the expansion,maintenance,and metabolic profiling of functional HSPC.The optimized expansion strategy for HSPC based on signaling pathway regulation was verified.These results established the basis for elucidating the mechanism of Wnt,MEK/ERK and NF-κB signaling pathways in regulating the biological characteristics of HSPC.A better understanding concerning the signaling pathway during HSPCs culture process provides a potential pipeline for developing new strategies to promote HSPC expansion for clinical application. |
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