Alpha Lipoic Acid Promotes Development Of Hematopoietic Progenitor Derived From Human Embryonic Stem Cells By Antagonizing ROS Signals

To obtain enough amount of hematopoietic stem/progenitor cells(HSPCs)for clinical treatment is one of the grand goals for current research on hematopoiesis.In vitro hematopoietic differentiation of human pluripotent stem cells(hPSCs)is an important approach to produce HSPCs.At present,the methods to induce in vitro hematopoietic differentiation from hPSCs include co-culture of hPSCs with stromal cells(AGM-S3 and OP9 cell lines)and stromal-free cultures(monolayer differentiation culture or embryoid body).Factors affecting the efficiency of in vitro hematopoietic differentiation are related with external microenvironment and internal molecular regulation.The former involves stromal cells types and culture medium components,while the latter is related to gene express.Reactive oxygen species(ROS)levels affect various physiological processes of stem cell.Regulation of ROS plays an important role in the hematopoietic differentiation of stem cells.Small molecules compounds that can decrease ROS level and inhibit cell apoptosis orautophagy,such as alpha lipoic acid(ALA),are useful for the maintenance or expansion of HSPCs.Base on this background information,we propose that ALA may have a regulatory role in the process of hematopoietic differentiation from hPSCs in vitro.Base on the in vitro hematopoietic differentiation system of co-culturing human embryonic stem cells(hESCs)and AGM-S3 stromal cells,we analyzed the effect of ALA on the in vitro hematopoietic differentiation efficiency of hESCs by adding ALA into hematopoietic inducing medium.We used flow cytometry to analyze hemogenic endothelial cells(HECs)and HSPCs at different time points.Colony-forming unit(CFU)assay and liquid differentiation were used to analyze the effects of ALA on the differentiation potential of HSPCs.The results indicated that ALA could significantly promote the production of HECs and HSPCs derived from hESCs.To decipher the molecular mechanism of ALA improving the efficiency of hematopoietic differentiation,we sorted HECs at day 8 of hematopoietic differentiation for transcriptome analysis.GO enrichment analysis and GSEA analysis were used to analysis the pathways or signals changed by ALA.The results showed that ALA upregulated the endothelial-to-hematopoietic transition(EHT)by positively expressing RUNX1,GFI1,GFI1B,MEIS2,HIF1A and downregulating the EHT negative genes SOX1 7,TGFB1,TGFB2,TGFB3,TGFBR1,TGFBR2.ALA also upregulated ROS-regulated genes including HIF1A,FOXO1,FOXO3,ATM,PETEN,SIRT1 and SIRT3,We found that ALA did not change the differentiation potential of HSPCs in late stage by CFU analysis;at the same time,ALA reduced the ROS level and inhibited the apoptosis of HSPCs.In addition,ALA can increase the proportion and absolute number of CD34+CD38-hematopoietic progenitor cells derived from cord blood mononuclear cells with 7 days in vitro culture,and can improve the rate of peripheral blood chimerism in immunodeficient mice by retro-orbital venous sinus transplantations.In conclusion,ALA plays an important role in increasing the production of hPSCs derived HSPCs and maintain the function of HSPCs,providing a new perspective for understanding the early hematopoiesis process.