Droplet Microarray Based Screening Identifies Proteins for Maintaining Pluripotency of hiPSCs.

Abstract

Human induced pluripotent stem cells (hiPSCs) are crucial for disease modeling, drug discovery, and personalized medicine. Animal-derived materials hinderapplications of hiPSCs in medical fields. Thus, novel and well-defined substrate coatings capable of maintaining hiPSC pluripotency are important for advancing biomedical applications of hiPSCs. Here a miniaturized droplet microarray (DMA) platform to investigate 11 well-defined proteins, their 55 binary and 165 ternary combinations for their ability to maintainpluripotency of hiPSCs when applied as a surface coating, is used. Using this screening approach, ten protein group coatings are identified, which promote significantly higher NANOG expression of hiPSCs in comparison with Matrigel coating. With two of the identified coatings, long-term pluripotency maintenance of hiPSCs and subsequent differentiation into three germ layers are achieved. Compared with conventional high-throughput screening (HTS) in 96-well plates, the DMA platform uses only 83 µL of protein solution (0.83 µg total protein) and only ≈2.8 × 10⁵ cells, decreasing the amount of proteins and cells ≈860 and 25-fold, respectively. The identified proteins will be essential for research and applications using hiPSCs, while the DMA platform demonstrates great potential for miniaturized HTS of scarce cells or expensive materials such as recombinant proteins.