Dataset Information

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

ABSTRACT:

Background

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.

Methods

We established a monolayer, chemically defined culture system to induce hematopoietic differentiation from hPSCs in 8 days.

Results

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.

Conclusion

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.

SUBMITTER: Duan F

PROVIDER: S-EPMC6062919 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe production of cardiomyocytes from human induced pluripotent stem cells (hiPSC) holds great promise for patient-specific cardiotoxicity drug testing, disease modeling, and cardiac regeneration. However, existing protocols for the differentiation of hiPSC to the cardiac lineage are inefficient and highly variable. We describe a highly efficient system for differentiation of human embryonic stem cells (hESC) and hiPSC to the cardiac lineage. This system eliminated the variability in cardiac differentiation capacity of a variety of human pluripotent stem cells (hPSC), including hiPSC generated from CD34(+) cord blood using non-viral, non-integrating methods.Methodology/principal findingsWe systematically and rigorously optimized >45 experimental variables to develop a universal cardiac differentiation system that produced contracting human embryoid bodies (hEB) with an improved efficiency of 94.7±2.4% in an accelerated nine days from four hESC and seven hiPSC lines tested, including hiPSC derived from neonatal CD34(+) cord blood and adult fibroblasts using non-integrating episomal plasmids. This cost-effective differentiation method employed forced aggregation hEB formation in a chemically defined medium, along with staged exposure to physiological (5%) oxygen, and optimized concentrations of mesodermal morphogens BMP4 and FGF2, polyvinyl alcohol, serum, and insulin. The contracting hEB derived using these methods were composed of high percentages (64-89%) of cardiac troponin I(+) cells that displayed ultrastructural properties of functional cardiomyocytes and uniform electrophysiological profiles responsive to cardioactive drugs.Conclusion/significanceThis efficient and cost-effective universal system for cardiac differentiation of hiPSC allows a potentially unlimited production of functional cardiomyocytes suitable for application to hPSC-based drug development, cardiac disease modeling, and the future generation of clinically-safe nonviral human cardiac cells for regenerative medicine.