LTB4 Promotes The Differentiation And Maturation Of Human Embryonic Stem Cells To Erythrocytes

Background:Red Blood Cells（RBCs）transfusion is a major therapeutic modality to improve anemia due to various causes and has important clinical applications.However,due to limited resources,short preservation period and the risk of transfusion-related disease transmission,the supply of RBCs is currently very tight at home and abroad,and there are various transfusion risks.The use of stem cells for clinical use is expected to become a new mode of blood supply and security in the future.As the starting seed cells for in vitro erythroid cell production,human Embryonic Stem Cells（h ESCs）have stronger self-renewal ability and triple germinal layer differentiation potential than adult hematopoietic stem cells,and are capable of large-scale expansion,making them ideal seed cells for obtaining a large number of erythroid cells.However,cultured Red Blood Cells（c RBCs）generated by in vitro induction of h ESCs are difficult to be applied as clinical blood products,and the biggest obstacle is that c RBCs are relatively large,have a low enucleation rate,and express Fetal Hemoglobin（Hb F）.Numerous studies have shown that c RBCs generated from human pluripotent stem cells in vitro lack adult hemoglobin（Hb A）expression,different from c RBCs from Peripheral Blood（PB）.Therefore,in order to explore these differences and the reasons for the functional immaturity of c RBCs from different sources for better use in the in vitro preparation of erythroblasts from pluripotent stem cells,we performed methylation microarray analysis of erythroblasts from different sources to analyze the reasons for the functional imperfection of c RBCs at the genetic level.The analysis identified potential impact molecule leukotriene B4（LTB4）receptors:LTB4R and LTB4R2.The molecule with the lowest differential P value and highest enrichment occurred simultaneously at the sites（Ery-DMS-h ESCs⁺/PB^-）where hypermethylation occurred in h ESCs-derived c RBCs（h ESCs-c RBCs）and hypomethylation occurred in PB-derived c RBCs（PB-c RBCs）.LTB4R and LTBR2 are receptors of LTB4,which is produced by arachidonic acid through multiple steps,and the pathway also produces other leukotriene isoforms such as LTC4 simultaneously.There are two other metabolic pathways of arachidonic acid.The metabolites of the cytochrome P₄₅₀ and cyclooxygenase（COX）pathways,hydroxyeicosatetraenoic acids（HETEs）and prostaglandin E₂（PGE₂）,have been reported in several papers to promote hematopoietic stem and progenitor cells（HSPCs）production and erythrocytes development.It has been shown that LTB4 promotes proliferation and differentiation of CD34⁺HSPCs derived from umbilical cord blood（UCB）to granulocytes and has anti-apoptotic effects.LTB4 and other leukotriene isoforms such as LTC4 and LTD4 also promote hematopoiesis in mice and zebrafish.In other studies on leukotriene regulation,LTB4was found to promote angiogenesis from mouse embryonic stem cells.And based on our comparison of differential methylation state and expression of LTB4 receptors in erythroblasts from different sources,we found that LTB4R and LTB4R2 were hypermethylated and lowly expressed in h ESCs-c RBCs and hypomethylated and highly expressed in PB-c RBCs.We hypothesized that this difference might be altered by adding LTB4 and subsequently promote the development and maturation of h ESCs-c RBCs.Objective:Based on the above analysis and the questions raised,this study aims to investigate in depth whether LTB4 promotes the differentiation and maturation of h ESCs to erythroblasts and the mechanism of its action,so as to optimize the red lineage cell induction protocol and promote the developmental maturation and function of h ESCs-c RBCs.Methods:To determine the key role of LTB4 in the regulation of erythroid development and maturation of h ESCs,firstly,the expansion efficiency of the h ESCs-c RBCs induction system was optimized by adjusting the cell inoculation density,and the expression changes of LTB4 receptor in the h ESCs-c RBCs induction system were observed by real-time quantitative PCR（RT-q PCR）experiments.Secondly,LTB4 or LTB4 metabolic pathway inhibitors were added at different stages of the induction protocol to determine the effect of LTB4 on the differentiation and maturation of erythrocytes and other related cell lineage.Cells were subjected to Flow Cytometry（FCM）,phenotypic analysis,hematopoietic colony formation assay,RT-q PCR,and Richter-Kimsa staining to fulfill the comparison of maturation levels from different induction conditions.Then,cellular RNA-seq analysis and bioinformatics prediction were used to determine the downstream signaling pathways affected by LTB4 after acting on its receptor.By screening differentially expressed genes,GO（Gene Ontology）,KEGG（Kyoto Encyclopedia of Genes and Genomes）and PPI（Protein-Protein Interaction Networks）analyses were performed on the screened differentially expressed genes to clarify the LTB4 action mechanism.Finally,LTB4 was added to the protocol for UCB-derived mononuclear cells（UCB-MNCs）to erythrocytes induction to verify the effect of LTB4 on erythrocytes differentiation and maturation.Cell lineage analysis of LTB4 functioning in UCB-derived c RBCs（UCB-c RBCs）was performed by adjusting the stage of LTB4 supplement.Cells were subjected to FCM,phenotypic analysis,RT-q PCR,and Richter-Kimsa staining to fulfill the comparison of maturation levels.Results:In this study,the h ESCs were first cultured and amplified in a feeder-free culture protocol,and the cells expressed high levels of pluripotent stem cell genes and proteins such as SSEA4 and TRA-1-60.This indicates that these h ESCs cultured in vitro have good pluripotency and can be stably passaged.We used two densities（2×10⁵/well and 6×10⁵/well）to inoculate cells and successfully established and optimized a staged induction protocol to induce differentiation of h ESCs to c RBCs using a 3D Embryoid Bodies（EBs）induction system.Inverted stage contrast microscopy was used to observe the gradual enlargement and vacuole formation of h ESCs into EBs in the 3D culture protocol used in this study,followed by the gradual release of a large number of individual suspension cells with strong refractive index.The proportion of low-density CD43^-CD45^-CD34⁺CD144⁺Hemogenic Endothelia Cells（HECs）was higher than high-density judged by FCM and gradually decreased after reaching a peak of 21%on Day 6 of the induction protocol.The proportion of low-density CD34⁺CD45⁺HSPCs and CD34^-CD45⁺mature HSPCs（m-HSPCs）was higher than that of high-density and gradually decreased after reaching a peak of 23%in the induction differentiation system Day 12.The percentage of low-density CD71⁺CD235a⁺erythrocytes was higher than high-density in the early stages,and eventually both increased to more than 90%.Mitochondrial membrane potential assay and hematopoietic colony formation assay showed that low-density culture generated hematopoietic cells with good metabolic status and significantly promoted colony growth,especially resulted in a significant increase in the proportion of BFU-Es which representing early erythrocytes.The hemoglobin content analysis showed that the hemoglobin content of the cells at Day 12 of low-density was higher than that of high-density,and there was no significant difference in the hemoglobin content of c RBCs obtained at Day 18,but there was a significant increase in the amplification fold of low-density c RBCs.Based on the optimized induction protocol,the expression of LTB4receptors at low-density was detected.The results of RT-q PCR showed that the relative expression of LTB4 receptor gradually increased with the induction time and was highly expressed in hematopoietic cells,suggesting that LTB4 and its receptor may play a role in erythrocyte development.We explored LTB4’s effect on the differentiation of h ESCs to mesoderm cells,HECs,HSPCs and c RBCs by staged addition of 50 n M LTB4 or 50μM Zileuton,a 5-lipoxygenase inhibitor that blocks the intact LTB4 metabolic pathway.Based on the 4-stage induction protocol,the supplement of LTB4 or Zileuton in each stage was marked as S1 to S4 respectively.The FCM assay showed that additions of LTB4 in S1 and S2both resulted in higher CD43^-CD45^-CD34⁺CD144⁺HECs on Day 6 than the control,and the HECs from 2nd stage additions of Zileuton were lower than the control.All groups added LTB4 derived CD34⁺CD45⁺HSPCs and CD34^-CD45⁺m-HSPCs at a higher percentage on Day 12 than control,and zileuton treatment came out CD34^-CD45⁺m-HSPCs at a lower percentage than control.The percentage of CD71⁺CD235a⁺erythrocytes in the experimental and control groups gradually increased to more than90%.Meanwhile on Day 6,the percentage of BFU-Es,which representing developmentally early erythrocytes,from in the 1st and 2nd stages of LTB4 addition was higher than that from the control,where the number of hematopoietic colonies from the 2nd stage of LTB4 addition was significantly higher than other groups.The percentage of c RBCs obtained by induction was above 90%in both experimental and control groups.Of note,the addition of LTB4 in the 1st,2nd and 4th stage leaded to higher amplification fold of c RBCs than the control,and the addition of Zileuton in the2nd stage resulted in less number of erythrocytes than the control.The relative expression ofβ-globin was higher in c RBCs from groups with LTB4 added in the 2nd and 4th stage than in the control,and the addition of Zileuton in the 2nd and 4th stage had lowerβ-globin expression than the control.Meanwhile,the enucleation rate of c RBCs from groups with the addition of LTB4 at the 2nd and 4th stage was higher than that from the control group,suggesting that LTB4 plays a positive regulatory role in the differentiation of h ESCs to erythrocytes.Following the initial clarification that LTB4 regulates the differentiation of h ESCs into erythrocytes,to explore the molecular mechanism of its regulation of hematopoietic differentiation,we further sequenced the transcriptome of cells at the sixth day of induction with LTB4 addition in the early stage of hematopoietic differentiation by MGIseq2000 sequencing platform and DNBseq G400 sequencing platform.Combined with the sequencing data,the differentially expressed genes were analyzed by matching.The results showed that the total number of differential genes was 1012 from the comparation of the 1st stage LTB4 addition and the control cells,and 991 from the comparation of the 2nd stage of adding LTB4 and the control cells.The results of differential gene KEGG enrichment analysis showed that the different genes between LTB4 and control groups in the 1st stage were mainly enriched in hematopoietic-related regulatory signals such as Calcium signaling,MAPK,PI3K-Akt,Rap1,Ras,and Wnt;the different genes between LTB4 and control groups in the 2nd stage were mainly enriched in hematopoietic-related regulatory signals such as arachidonic acid metabolism,NF-κB,renin-angiotensin system,and thyroid hormone synthesis.The bioinformatic analysis provides an important basis for our preliminary study on the mechanism of the effect of LTB4 on hematopoietic differentiation.In the above study,we unexpectedly found that LTB4 promoted the enucleation of h ESCs-c RBCs.To verify the effect,we added 50 n M of LTB4 to the induction system of UCB-c RBCs by each stage.The results showed that LTB4 could promote the amplification of UCB-derived HSPCs and c RBCs,and the rate of enucleation of UCB-c RBCs was significantly increased.However,there was no significant effect on the differentiation ratio of UCB-derived HSPCs and c RBCs,and the expression level ofβ-globin in UCB-c RBCs.The above findings confirm that LTB4 can indeed promote the enucleation of c RBCs.Conclusion:In summary,this study successfully optimized a 3D EBs culture protocol for the directed induction of h ESCs differentiate to erythrocytes using different cell inoculation densities.Highly CD235a-expressing c RBCs were successfully obtained on Day 18 of induction.An examination of the role of LTB4 and its metabolic pathway inhibitors in the differentiation of h ESCs to erythrocytes was performed.The role of LTB4 and metabolic pathway inhibitors in the differentiation of h ESCs towards erythrocytes was initially examined.LTB4 was found to promote the differentiation of h ESCs towards mesoderm cells,HECs,HSPCs and c RBCs.LTB4 showed an overall positive regulation of h ESCs-c RBCs differentiation at different stages.And its denudative effect was verified in the induction system of UCB-c RBCs.The mechanism of action of LTB4 may be related to signaling pathways such as Rap1,PI3K-Akt and Wnt.And the mechanism of its action in the second phase may be related to signaling pathways such as arachidonic acid metabolism,NF-κB,and thyroid hormone synthesis.This study will provide the necessary theoretical basis and technical support for our subsequent in-depth exploration of the mechanism of the role of LTB4 in the developmental maturation of h ESCs into erythrocytes,the construction of an optimized system for the targeted induction of h ESCs into erythrocytes,and the efficient preparation of h ESC-c RBCs.