Tracking Pluripotent Stem Cell Differentiation

A modified H9 pluripotent stem cell line where eGFP was expressed under the control of LMX1A promoters (LMX1A-eGFP) (Niclis et al., 2017 (link)) was used to track the expression of LMX1A. All plates or flasks were pre-coated with 0.5 µg/cm² Laminin-521 (Life Technologies, Australia). Undifferentiated cells were grown using a slightly modified method of (Watmuff et al., 2015 (link)). Briefly, pluripotent stem cells were seeded at a density of 3,000 cells per cm² and cultivated in Essential 8 Medium with Essential 8 supplement (Thermo Fisher Scientific, Australia) and Penicillin/Streptomycin (Life Technologies, Australia) at 37°C in a humidified incubator containing 5% CO₂. Cells were passaged at ∼80% confluence (ReLeSR™, Stemcell technologies, Australia). To enhance the survival of cells seeded at low-density, the ROCK inhibitor (10 μM Y-27632) was added to the medium for the first 24 h after passaging. Cells were maintained for a maximum 10 passages.