NOTCH Signaling Of The Vascular Niche Prompt The Differentiation Of Functional HSCs From HPSCs

Hematopoietic stem cells(HSCs)are a class of pluripotent stem cells from hematopoietic tissue that has the ability of self-renewal and differentiation potential to all types of mature blood cells.HSCs are the ideal seed cells for a mature blood cell that is available for clinical hematopoietic stem cell transplantation and blood transfusion therapy.However,there are many limits for clinical application such as the shortage of hematopoietic stem cells,the low success rate of matching,and the high cost of transplanting.To meet these problems,human induced pluripotent stem cells(hiPSCs)were employed for producing HSCs.Nevertheless,the HSCs induced by hiPSCs only have the phenotype of hematopoietic stem cells,but short of the ability of long-term reconstruction of hematopoiesis as in vivo.The main reason is that the complex individual hematopoietic development is not fully understand,hematopoietic differentiation system established in vitro cannot stimulate hematopoiesis development environment in vivo,and the differentiation system need to be further optimized.The present research indicates that the key step in the development of hematopoietic stem cells is the differentiation process of hematopoietic endothelial cells into both hematopoietic cells and endothelial cells.During the process of human hematopoiesis,NOTCH signaling pathway has been proved to play a key role in regulating the differentiation and proliferation of hematopoietic endothelial cells into hematopoietic cells.Additionally,the expression of DLL4 ligand in NOTCH signaling pathway is one of the main elements.DLL4 overexpressed endothelial progenitor cells depressed the expression of DLL4 and promote differentiation of negative hematopoietic endothelial cells into hematopoietic stem cells,which still short of the ability of long-term reconstruction of hematopoiesis.All these results reveals that there is other important elements participate in the production of HSCs.The activation of the NOTCH signaling pathway mediated by the Vascular Niche and the expression of the downstream hematopoietic transcription factor,runx1 and gata-2,are critical to the generation of hematopoietic stem cells.Based on the above analysis,this study is to generate hemogenic endothelial cells from human induced pluripotent stem cell(hi PSC)in vitro.Afterwards,the hemogenic endothelial cells are co-cultured with the vascular niche cells.The vascular niche was thus formed and promotes hemogenic endothelial cells further differentiation to HSCs.The function in vitro and in vivo of HSCs are proceed to further understanding of the mechanism of vascular niche and NOTCH signaling pathway in affecting hematopoietic stem cells production and self-renewal.Our research is divided into the following parts:Initially,the human embryonic stem cells H9 cell line was used to establish a hematopoietic differentiation system in vitro.We established a method in which the H9 cell line was co-cultured with MEF in a single cell growth state.Using this growth characteristic of the H9 cell line,we established and optimized the induced differentiation system of spin EB.Through this induced differentiation system,the H9 cell line was subjected to hematopoietic induction in vitro.It was detected by flow cytometry,on the 12 th day after differentiation,the expression lof CD34 is about 15%,and the CD45 is about 10%,which can efficiently induce the differentiation of H9 cells into hematopoietic stem cells.After proving the feasibility of this spin EB's induced differentiation system,single cell cultured hiPSCs with reporter gene runx1c(runx1c-hiPSCs cells)were differentiated by spin EB method.We detected the surface markers of differentiated cells by flow cytometry on the 3rd,6th,9th,and 13 th days of differentiation,and found that CD34+ cells reached 11% on the 9th day of differentiation,and CD45+ cells reached 15% on the 13 th day of differentiation.The CD34 positive cells were sorted by MACS Micro Beads at day 10 after differentiation,and the percentage of CD34 positive cells after sorting was more than 90% by flow cytometry.The sorting cells were able to differentiate into endothelial cells and hematopoietic cells in vitro.runx1c-hiPSCs cells expression GFP green fluorescence,and td Tomato orange fluorescence will appear along with the expression of runx1 c during the Induced differentiation process.The cells with bifluorescence markers were able to generate the cells at different stages of differentiation,and were beneficial to the tracing and detection of cells after transplantation in vivo.To further understanding of the mechanism of the regulation role of vascular niche in affecting hematopoietic stem cells production,we established an vasucar niche in vitro using the human umbilical vein endothelial cell line,Vera Vec,which transfected with E4ORF1.We established a model of the vascular niche to promote endothelial hematopoiesis in vitro through use Vera Vec cells co-culture with CD34+ cells,and preliminarily explored the mechanism of endothelial microenvironment and NOTCH signaling pathway promoting hematopoietic transcription.Flow cytometry showed that Vera Vec cells only expressed endothelial cell markers CD31,CD144,and KDR,and Western blot and q RT-PCR detected the expression of ligands and receptors of NOTCH signaling pathway in Vera Vec and HUVEC cells.In Vera Vec cells,the expression of the ligand DLL4 of the NOTCH signaling pathway was significantly higher than that of HUVEC(primary cell line).Therefore,Vera Vec cells are more ideal co-culture system cells than HUVEC cells and can help establish an vascular niche that highly expresses DLL4.Afterwards,transfected Vera Vec cells were co-cultured with CD34 positive hemogenic endothelial cells.Whereafter,the expression of runx1 c and further HSCs production was increased during the hiPSCs differentiation period.In this study,a method of inducing hiPSCs differentiation by spin EB was established and optimized,which could enrich hemogenic endothelial cells.Through the co-culture of CD34 positive hemogenic endothelial cells and DLL4 high expressed vascular niche cells Vera Vec,an vascular niche was formed and proved promotes hematopoietic differentiation from human pluripotent stem cells.This model can be applied to study the mechanism of vascular niche to promote hematopoietic differentiation from human pluripotent stem cells.Furthermore,this model lay a foundation for the production of hematopoietic stem cells from hiPSCs.