Research Associate Cincinnati Children's Hospital Medical Center Cincinnati, Ohio, United States
Abstract: Organoid technology has flourished due to its ability to mimic human development, making it an invaluable tool for investigating the causes of diseases. The "in-a-dish" cellotype-phenotype association strategy is a powerful approach for studying how donor-specific genetic traits influence phenotypes within a cell-type specific regulatory context. En masse phenotyping, combined with donor pooling techniques, offers valuable insights into cellotype-phenotype associations under controlled exposures. This methodology is key to discovering biomarkers and therapeutic targets for precision medicine aimed at heterogeneous metabolic diseases. We have applied donor pooling and liver organoid technology to develop a multiple-donor Population Organoid Panel (PoP) platform. Using this model, we simultaneously analyzed cellotype-phenotype associations in MASLD and explored cell-type-specific responses. We induced PoPs from 25 donors and established a MASLD model, followed by single-omics analysis. Employing the Demuxlet algorithm for multiplexed scRNA-seq, we achieved a high accuracy of 97% to 99% in identifying singlet donors. This level of accuracy is crucial for ensuring that cells are correctly assigned to their respective donors, which is essential for downstream analyses. Additionally, a correlation study between lipid accumulation phenotypes and multiplexed scRNA-seq revealed a link between fat accumulation and the expression of genes associated with steatosis. In hepatic and stellate cell populations, genes related to the liver X receptor (LXR) were found to correlate with inflammatory responses and steatosis. We also induced fibrosis in HLO and assessed it by measuring the liver fibrosis marker M2BPGi and cytotoxicity using CK18f levels. Results showed that M2BPGi secretion increased 1.75-fold (p < 0.05) with treatment using low concentrations of oleic acid. When glucose and insulin levels were reduced by a factor of four, M2BPGi secretion decreased 0.37-fold (p < 0.05). The single-cell genomics approach, when combined with PoP, enables highly accurate downstream analysis of each donor’s characteristics. These analyses allow for an investigation of the effects of environmental changes on different cell types and pathways, all while considering the genetic background of each donor.
