Amplification Of Hematopoietic Stem Cells By In Vitro Regulation Of Wnt Signaling Pathways

Hematopoietic stem cells are adult stem cells, capable of self-renewal and lifelong supplement including differentiation into all hematopoietic lineages including erythrocytes, neutrophils, basophils, eosinophils, lymphocytes monocytes macrophage and platelets.Hematopoietic stem cell transplantation as an effective means of clinical treatment of leukemia and aplastic anemia refractory hematological disorders and other tissue cancer has been widely used. Currently shortage of hematopoietic stem cells is to limit such treatment bottleneck. In theory, the ex vivo expansion of hematopoietic stem cells is to solve this problem, the most direct and effective way. However, due to lack of effective culture system, in vitro amplification of HSC still far from applicating the clinical treatment. To solve the clinical treatment of hematopoietic stem cells severe shortage of difficulties, many domestic and foreign laboratories put a lot of manpower and material resources, trying to hematopoietic stem cells amplification strategy in vitro. Now that the hematopoietic stem cell self-renewal and expansion of the comprehensive results by a variety of factors stimulated. Studies have shown that hematopoietic stem cells in vitro amplification and function maintenance culture system is affected by many factors cytokines, serum and oxygen content. Wnt signaling, pathway widespread in and highly conserved from invertebrate to vertebrate. It involve in at least three different cell signaling pathways:the classical Wnt/β-catenin signaling pathway and non-canonical Wnt/calcium signaling and Wnt/JNK signaling pathway. Previously studies have shown that, Wnt/βcatenin signaling pathway can regulate hematopoietic stem cell self-renewal and expansion, but the relevant research results are not consistent and sometimes conflicting. We speculate that periodically correct dose Wnt/β-catenin signal may be key to promoting hematopoietic stem cell expansion. In this article, we have adopted advanced technology of controlled genetically, to study the impact of the signal amplification of hematopoietic stem cells by regulating Wnt/β-catenin activation of the signal. Hematopoietic stem and progenitor cells be introduced into a gene regulating active βcatenin-ΔNβ-catenin-ER, the activation of the gene is responed to exogenous estrogen 4-OHT,β-catenin activity and 4-OHT have a dose-response relationship. Through the culture system with different concentrations of 4-OHT, can regulate Wnt/β-catenin signaling activation degree in hematopoietic stem/progenitor cells, which would help us to be able to studies the effects of different β-catenin level activation of hematopoietic stem cells amplification and identify the optimal dose to provoke hematopoietic stem cells expansion in vitro.First we viral transduction method is optimized so that viral transduction efficiency to 50% or more,also found that serum-free culture conditions do not reduce viral transduction efficiency of hematopoietic stem and progenitor cells. We activate the in the use of four concentrations of 4-OHT inductionwas found after 0.5μM,10μM,1.5μM doses of 4-OHT induced 9days significant increase in the number of hematopoietic stem cells, which 1.0μM and 1.5μM doses of 4-OHT treatment is better, the total number of LSK cells relative to vehicle control group (0μM) were about 2-fold amplification.But we treated the 40HT under serum-free culture conditions found,0.5μM 1.0μM group significant increase number of LSK cells,1.5μM dose group cell death,0.5μM dose treatment groups, which since the strategy of passage we used caused by different.Our Lab used RNAarray techniques analyze RNA transcripts of stromal cells during hematopoietic recovery, identified a series of proteins such as IGFBP-2, RSPO-2 and RSPO-3 which likely contribute to promote hematopoietic stem cells expansion. We use RSPO family members to regulate Wnt signaling in hematopoietic stem cells, study its effect on hematopoietic stem cell expansion. Wnt3a+RSPO-3 and Wnt5a+ROSP-2 increased the number of LSK more than 1.5 times compare with the control.