Biphasic modulation of insulin signaling enables highly efficient hematopoietic differentiation from human pluripotent stem cells

Fuyu Duan, Rujin Huang, Fengzhi Zhang, Yonglin Zhu, Lin Wang, Xia Chen, Lufeng Bai, Wei Guo, Sophia Chia-Ning Chang, Xiaoyu Hu, Jie Na

Hematopoietic lineage cells derived from human pluripotent stem cells (hPSCs) hold great promise for the treatment of hematological diseases and providing sufficient cells for immune therapy. However, a simple, cost-effective method to generate large quantities of hematopoietic stem/progenitor cells (HSPCs) is not yet available. We established a monolayer, chemically defined culture system to induce hematopoietic differentiation from hPSCs in 8 days. We found that insulin-free medium allowed hPSCs to leave pluripotency promptly and preferably enter the vascular lineage. Addition of insulin during the later stage of differentiation was essential for the efficient induction of hemogenic endothelium and the emergence of large numbers of CD34+CD43+ HSPCs, while no insulin condition preferably permits endothelial differentiation. Global transcriptome profiling revealed that HSPCs differentiated using our protocol were similar to embryoid body-derived HSPCs. HSPCs obtained from our differentiation system formed robust erythroid, granulocyte and monocyte/macrophage colonies in CFU assay, and can be induced to generate functional macrophages with robust phagocytic ability. Our results demonstrated that proper manipulation of insulin-mTOR signaling can greatly facilitate HSPC formation. This finding can be further exploited to formulate cost-effective differentiation medium to generate large quantities of cells of desired blood lineages for regenerative medicine.