| Background:During mammalian embryonic development,hematopoiesis could be classified to primitive hematopoiesis(originate from the yolk sac,YS)and definitive hematopoiesis(originate from aorta/gonad/mesonephros,AGM).Mechanism research is of great significance for understanding human hematopoietic processes and related clinical applications.The invention of human embryonic stem cell culture technology and adult cell reprogramming technology makes human pluripotent stem cell(hPSC),including human embryonic stem cell(hESC)and human induced pluripotent stem cell(hiPSC),become important starting cellular sources for in vitro culture system.Recent years in vitro co-culture methods for blood cell differentiation have made significant progress so as to obtain a relatively large number of red blood cells and other types of blood cells from hESC/hiPSC.These systems not only provide the possibility of producing blood cells in vitro,but also provides a good model for studying the molecular regulation mechanism of hematopoietic differentiation.The human RUNX1 gene is 216 kb in length,locates at human chromosome 21(21q22.12),and has two promoters,PI(distal)and P2(proximal).It is the most important regulatory gene for hematopoiesis,especially in definitive hematopoiesis.RUNX1 has a lot of splicing isoforms,such as RUNX1a,RUNX1b,RUNX1c and RUNX1-205.The RUNXlb inducible hES cell line had been established in our laboratory.Overexpression of RUNXlb from early stage blocked hematopoiesis,which could be rescued by RepSox,a specific inhibitor of TGF-? signaling pathway.The molecular mechanism of blockage effect of RUNX1b on hematopoiesis needs further exploration.Except for RUNXla/b/c,RUNX1-205,a novel splicing variant of human RUNX1 rarely studied,encodes the longest protein with the complete coding region.RUNX1-205 only lacks exon 6 in comparison with RUNXlb.A homologous gene of RUNX1-205 in mice,Runx1-202,has complex functions in hematopoiesis,Indicating that RUNX1-205 plays an important role in human hematopoiesis.However,the function of RUNX1-205 on human hematopoiesis has not been explored using an in vitro system.It is of great significance to elucidate the molecular mechanisms of different RUNX1 splicing isoforms that regulate the generation of hematopoietic cells from hESC.Blood circulation disorder or obstruction was named as blood stasis syndrome(BSS)in traditional Chinese medicine(TCM),which might be caused by obstacle anemia,abnormal blood circulation and microcirculation,vascular endothelial lesion and blood composition changes.Blood-activating Chinese medicine,such as Spatholobus suberectus Dunn,could efficiently improve hematopoiesis and treat BSS.Therefore,we try to use AGM-S3 co-culture system to investigate phenolic compounds of Spatholobus suberectus Dunn and elucidate the cellular and molecular mechanism of their efficacies.Methods:The experiments were performed based on piggyBac eukaryotic inducible system,PB-tet-on-OE,and hESC/AGM-S3 co-culture in vitro hematopoiesis system.Cellular biological techniques,such as fluorescence-activated cell sorting(FACS),hematopoietic colony assay,May-Grunwald-Giemsa staining(MGG),embryoid body formation(EBs),and molecular biological techniques,such as Western blot(WB),Quantitative Real Time Polymerase Chain Reaction(qRT-PCR),were applied to elucidate the molecular/cellular mechanisms of RUNX1(RUNX1b and RUNX1-205)on hematopoiesis.And at different stage the four single compounds of Spatholobus suberectus Dunn were added into co-culture system at different final concentration to detect their influence on hematopoietic progenitors and erythroid progenitors.Results:1.Based on H1 hESC the CDKN2B(P15),CDKN2C(P18)or CDKN1A(P21)inducible hES cell lines were established,which pluripotency is normal.Overexpression of RUNX1b and up-regulation of TGF-? signaling pathways from early stage blocked hematopoiesis,which might be associated with cell cycle status change,such as G1 arrest.Cell cycle-related genes,for example,P15,P18,and P21.were up-regulated with overexpression of RUNX1b,and restored to original level when TGF-P signaling inhibitor(0.33 ?M RepSox)was added.When different DOX concentration was applied to induce RUNX1b from D0,the expression level of P15,P18,and P21 show a close relative to RUNXlb at D4.Overexpression of PI5,P18,and P21 from DO and directly added TGF-?1 to RUNX1b/AGM-S3 co-culture from DO,which all reduced the production of CD34+CD43+ and CD34+CD45+ populations,showing hematopoiesis blockage,but CD34+ population was not significantly influenced.2.Based on H1 hESC the RUNX1-205 inducible hES cell lines were established,which pluripotency is normal.Alignments of amino acid sequences of RUNX1 homologous genes in evolution revealed that splicing variants homologous to RUNX1-205 have kept highly conservation during evolution.Overexpression of RUNX1-205 from D0-D2 showed blockage effect on hematopoiesis,but when it was induced from D6 or later,the effect became weaker or even disappeared.Both in co-cultures with AGM-S3 cells and during EBs formation,induced RUNX1-205 overexpression from early stage down-regulated the expression of hematopoiesis-related genes.At D14 co-cultured cells induced RUNX1-205 overexpression from DO were significantly blocked the formation of CFU-E,BFU-E,CFU-Mix and CFU-GM colonies in hematopoietic colony assay.However,the numbers of colonies were restored to normal level when it was induced from D6.In transgenic 293T cells,RUNX1-205 is much more stable than RUNXlb.In H1 hESCs co-cultured with AGM-S3 cells,mRNA expression of RUNX1-205 was higher at the early stage(D2-D4),lower at the late stage(after D4),mRNA expression of RUNXlb was higher at the early stage(D2-D4),lowest at D6,and gradually increased at the late stage(after D8).3.H1 hESCs co-cultured with AGM-S3 cells was added catechin at 10 ?M from D12,the proliferation and differentiation of hematopoietic progenitors and erythroid progenitors will be obviously increased.Compared with the control group(equal volume of DMSO),CD34+CD45+cells were increased 2.5-fold(P<0.0001),and GPA+CD71+cells were increased 1.5-fold(P<0.0001);under the same conditions,the co-cultured cells were stimulated by catechin to produce more CFU-E,BFU-E,CFU-Mix,and CFU-GM colonies in hematopoietic colony assay;meanwhile,qRT-PCR indicated that some important hematopoiesis and erythroid related genes were up-regulated after catechin was added.Conclusion:1.Overexpression of RUNX1b might enhance TGF-? signaling pathway,and then up-regulate the expression of P15,P18,and P21.It might result in G1 arrest of cell cycle,which probably lead to hematopoiesis blockage.Overexpression of P15,P18,and P21 from an early stage or up-regulated TGF-? signaling pathway,could block hematopoiesis,which is similar to the effect of RUNXlb.CD34+CD43+and CD34+CD45+populations were lost,but CD34+population was not significantly influenced.2.Overexpression of RUNX1-205 from an early stage(especially from DO)did not block induction of mesoderm,but blocked hematopoiesis and product of CD34+cells at an early stage,which led to loss of CD34+CD43+and CD34+CD45+populations at a late stage.At the same time,the expression of many important hematopoiesis-related genes were down-regulated,but when RUNX1-205 was induced from D6 or later,the effect became weaker or even disappeared,which is similar to the effect of RUNX1b.RUNX1-205 and RUNX1b have obvious difference in protein stability and expression profile during hematopoiesis.These results suggest that these two isoforms have similar functions and RUNX1-205 might play a complementary role to RUNXlb.3.FACS analysis,hematopoietic colony assay and qRT-PCR indicated that hematopoietic progenitors and erythroid progenitors could be significantly improved by catechin only using optimal concentration at a late stage of hematopoiesis.It indicated that the AGM-S3 co-culture system could be applied as an effective tool to screen the active compounds of TCM that promote the hematopoiesis,which have important potential application on clinical research and pharmaceutical industry. |
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