| Objective:Hematopoietic stem cells (HSCs), which could build up the whole blood system through self-renewal and multi-lineage differentiation, are the only cell sources that are used into clinical treatment currently. Although hematopoietic stem cells transplantation (HSCT) has already been the routine therapy in treating hematological malignancies, lack of HLA-matched donor greatly limited the usage of HSCs clinically. As a new source of HSCs, umbilical cord blood has various advantages, such as tolerance of partially HLA-mismatch and low incidence of GVHD, et al. However, the low number of HSCs in a single cord blood sample makes it difficult for HSCT in adult patients. Entering cell cycle is the last step of HSCs self-renewal and ex vivo expansion, while manipulation in the G1 phase might be a key step during the self-renewal progress. Therefore, for the sake of clinical treatment demand and the study of stem cells expansion and the regulation mechanism of self-renewal, we chose cell cycle regulation key protein, p18, as the target to design and screen small molecule compounds to expand HSCs during ex vivo culture and explored the regulated mechanism.Methods:We first knocked down the expression of p18 in human umbilical cord blood using lentivirus infection and then analyzed stem cell markers and hematopoietic capacity changes. After that we designed and screened a series of small molecule inhibitors against p18 through computer aided drug screening and tested the mouse HSCs ex vivo expansion effect of those compounds. When confirming the HSCs expansion effects, we further tested those compounds upon human umbilical cord blood HSCs and got a best hit compound,005A. Next we conducted dose response assay of 005A to optimized its HSCs expansion effect and ex vivo short-term and long-term hematopoietic capacity assays and xenograft assay into immunodeficient mouse..We also employed SPR assay and CDK6 kinase activity assay to confirm the interaction between 005A and p18. In order to explore the internal mechanism, we did RNA expression analysis using PCR technique.Results:The percentage and number of HSCs had a significant increase after p18 knock-down, so did the CFC number and the CAFC number. And part of our potential p18 small molecule inhibitors could increase the mouse HSCs expansion, especially the compound 005A with an ED50 of 5.2nM. The results of 005A upon human HSCs showed that 20nM 005A could significantly enhance the number of CD34+ CD49f+ cells. Meanwhile, the number of CFC and CAFC were also increased after 005 A treatment. The gold standard of hematopoiesis, xenograft assay into immnodeficient mouse, indicated that 005A could maintain cells’hematopoietic capacity after treatment and the success rate of transplantation was higher than untreated group. The protein binding assay and the protein function assay both showed that 005A could bind with p18 protein and inhibited its activity. Mechanism analysis suggested that Notch pathway was activated after 005A treatment.Conclusion:p18 knock-down assay showed that p18 was a critical protein and an ideal target in the regulation of HSCs expansion. Our newly discovered p18 small molecule inhibitor 005A showed promising effect upon HSCs expansion and sternness as well as hematopoietic capacity maintenance. The mechanism study indicated that 005A mainly activated Notch pathway to achieve its HSCs expansion effect. Moreover,005A treatment could enhance stem cell symmetric division but not accelerate cell cycle progress so that avoiding stem cell exhaustion. The above results suggested a novel small molecule compound 005A which could improve HSCs expansion and was promising for the future HSCT in clinical therapy. |
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