Institute of Zoology, Chinese Academy of Sciences (CAS), United States
Abstract: Pyrogens, including endotoxins and nonendotoxins, are among the key factors that impact the safety of parenterally applied drugs serve as contaminants. At present, conventional pyrogen detection methods are predominantly dependent on the Rabbit Pyrogen Test (RPT) and the Limulus Amebocyte Lysate (LAL) assay, both of which are animal-based. However, both methods have limitations: the RPT exhibits poor reproducibility, a high false-positive rate, and is cost-intensive; whereas the LAL test is restricted to detecting endotoxins and is constrained by the precipitous decline in the horseshoe crab population. The Monocyte Activation Test (MAT), based on monocytes, has become the most promising alternative to traditional pyrogen detection methods. However, the MAT is complex to operate, time-consuming, has significant individual differences, and usually requires a large amount of human blood, which greatly limits its application. Therefore, we have developed a rapid pyrogen detection method based on monocytes derived from human pluripotent stem cells (hPSCs) using a luciferase reporter system. Owing to the stability and pluripotency of hPSCs, we can obtain a large number of hPSC-derived monocytes (eMo) that are stable between batches and similar to peripheral blood monocytes through directed differentiation in vitro. We have discovered that eMo exhibits sensitive reporting capabilities for at least two types of pyrogens: lipopolysaccharide (LPS), a major component of the cell wall of Gram-negative bacteria, and lipoteichoic acid (LTA), a major component of the cell wall of Gram-positive bacteria. Further, we confirmed that eMo exhibits stable expression of TLRs, which are one of the main factors determining whether pyrogens can be stably detected. Thus, we have developed and validated a new pyrogen detection method based on the reporting system and directed differentiation of hPSCs in vitro, providing a new direction and approach for the replacement of traditional pyrogen detection methods.
Funding Source: Supported by the Strategic Priority Research Program of the Chinese Academy of Sciences，Grant No. XDC0200000.
