A method using ammonia to select hepatocytes from differentiated ones from pluripotent stem cells (iPSC, ESC)

Toxicology tests using hepatocytes have come to be widely used as an alternative way for testing drug toxicology using animals. The problem with using hepatocytes in toxicology tests is that it is difficult to supply large quantities of hepatocytes with the same characteristics. Therefore, a group from the National Institute of Child Health and Development proposes a method to create hepatocytes from pluripotent stem cells (iPSC, ESC) and enrich highly uniform hepatocellular populations from heterogeneous cell populations using ammonia.

After ESC is differentiated into hepatocytes, an ammonia treatment is performed for 2 days to select uniform hepatocytes that are resistant to ammonia toxicity. 70-80% will die at this stage. Surviving hepatocytes are cultured and proliferated on MEF-feeder. Hepatocytes selected in this way multiply about 30 times in 190 days. The detailed mechanism of ammonia toxicity is not known, but it is thought that ammonia ions compete with potassium ions and eventually die with intracellular and/or extracellular pH change. ALB, AFP, CYP3A4, CPS1, and OTC genes are highly expressed in ammonia-selected hepatocytes, and ALB and AFP decrease with the passage. CPS1 and OTC are genes involved in the metabolism of ammonia. CYP3A4 is one of the enzymes that metabolizes unwanted biological molecules.
The same is possible with hepatocytes made from iPSC.

The differentiated cells from iPSC and /or ECS were further cultivated and propagated in the ESTEM-HE medium (GlycoTechnica, Ltd.) .