R-spondin Protein Cooperates With Wnt Signaling Pathway To Promote The Self-renewal And Expansion Of Hematopoietic Stem Cells

Hematopoietic stem cell(HSC)is a kind of pluripotent stem cell,which has the ability of self-renewal and can differentiate into various types of blood cells.HSC transplantation has been widely used as an important treatment method for refractory hematological diseases.However,due to the lack of effective HSC amplification system in vitro,the shortage of HSC sources is an urgent problem to be solved in clinical treatment.HSC self-renewal and differentiation in vivo depend on the bone marrow microenvironment(Niche),which is considered to be the key to optimize in vitro culture and achieve successful HSC amplification.Wnt signaling pathway is one of the multiple signaling pathways involved in the self-renewal and maintenance of HSC in bone marrow Niche,and its activation degree is critical to the function of HSC.However,as an agonist of Wnt signaling,the role of R-spondin in the regulation of HSC self-renewal and expansion remains unclear.This study mainly investigated the effect of r-spondin protein in bone marrow Niche on the in vitro amplification of HSC in conjunction with the activation of Wnt signaling pathway.This article is divided into four parts :(1)the effects of RSPO2 and Wnt3 a on HSC amplification in vitro in serum-free co-culture system;(2)The effect of bone marrow stromal cells overexpressing RSPO2 on HSC amplification in vitro;(3)The effect of RSPO2 synergistic activation of Wnt/?-catenin signal on hematopoietic reconstruction in mice with HSC;(4)The regulation of RSPO2,Wnt3 a and Wnt5 a on HSC amplification in vitro.The results show that:1.RSPO2 can activate classical Wnt signaling pathway to regulate HSC amplification.We adopted serum-free in vitro co-culture method to make mouse bone marrow stromal cells OP9 cells and hematopoietic stem/ Progenitor cell(HSPC)contact co-culture,and add different working concentrations of RSPO2 and Wnt3 a cytokines.It was found that 20ng/ m L RSPO2 and 20ng/ml Wnt3 a could double the number of HSPC and long-term hematopoietic stem cells(LT-HSC)compared with the control group(without RSPO2 and Wnt3a).In addition,20ng/ m L RSPO2 and20ng/ m L Wnt3 a co-culture enhanced the self-renewal ability of HSC in vitro,which showed that the number of clones increased.The expression of receptor Tyrosine kinase(RET),one of the target genes of Wnt signaling in HSPC,was up-regulated by microcellular QRT-PCR.2.Overexpression of RSPO2 in stromal cells can also promote in vitro amplification of HSC and HSPC.We constructed an OP9 cell line that overexpressed RSPO2 by retrovirus infection and simulated the marrow Niche environment in vitro to explore the regulation of RSPO2 on HSC amplification.The results showed that overexpression of RSPO2 in stromal cells increased the number of HSC and HSPC by about 30% compared with the control group.3.HSPC of 20ng/ m L RSPO2 and 20ng/ m L Wnt3 a serum-free co-culture group were transplanted into recipient mice through competitive bone marrow transplantation.The hematopoietic reconstruction ability of HSPC after co-culture of20ng/ m L RSPO2 and 20ng/ m L Wnt3 a group was significantly enhanced compared with the control group,but the secondary transplantation results showed that the hematopoietic reconstruction ability of HSPC after co-culture of 20ng/ m L RSPO2 and 20ng/ m L Wnt3 a group was significantly decreased compared with the control group.HSC co-cultured with RSPO2 and Wnt3 a can enhance the potential of hematopoietic reconstruction in the short term.However,due to in vitro co-culture,HSC dryness has a certain loss,which can not maintain long-term hematopoiesis.4.In 20ng/ m L RSPO2 and 20ng/ m L Wnt3 a serum-free co-culture system groups,we added different concentration gradients of Wnt5 a to explore whether RSPO2 can simultaneously synergically activate non-classical Wnt signaling pathways to regulate HSC amplification.The results showed that adding 10ng/ m L Wnt5 a into 20ng/ m L RSPO2 and 20ng/ m L Wnt3 a serum-free co-culture group had the best amplification effect,and the number of amplified HSPC cells increased 2.4times compared with the control group.The number of HSC increased to 3 times that of the control group(without RSPO2,Wnt3 a,Wnt5a).In this study,the effect of RSPO2 and Wnt signaling pathway on in vitro HSC amplification and in vivo hematopoietic reconstruction was preliminatively discussed through in vitro simulated bone-marrow Niche culture HSC,providing reliable data for constructing an effective in vitro HSC amplification system and solving the problem of shortage of sources in clinical HSC transplantation treatment.It also provides a theoretical basis for studying the molecular mechanism of HSC self-renewal and multipotential differentiation.