Leveraging RNA-seq Deconvolution to Improve Complex In Vitro Model Characterization

Accelerating development of complex in vitro models (CIVMs) drives a need for additional methods to characterize these systems for use in toxicology and drug development. Relative to traditional cell culture, CIVMs may use a lower number of cells, are cultured for longer periods of time, and typically involve engineered 3-D cell organization. Standard single-cell assessment tools are not conducive to these types of complex models due to cell isolation stress, cell loss, and high cost. In this report, we benchmark RNA-seq deconvolution, which utilizes publicly available scRNA-seq datasets to predict cell proportions from bulk RNA-seq data derived from two CIVMs: a stem-cell-based human intestine epithelia model and a neonatal rodent testis model. We consider the impact of multiple imputation methods for scRNA-seq to restore the gene distribution of the original tissue to generate a better cell type signature and multiple deconvolution methods. The accuracy of deconvolution methods varied significantly in our analyses but provided valuable information on the emergence of an enterocyte cell population from the LGR5+ crypt stem cells following differentiation. In the testis model, deconvolution indicated that a small population of germ cells were retained over time, and that cell type estimates remained stable with physiologically relevant hormone stimulation. In our analysis, using imputed single cell references improved deconvolution accuracy. Deconvolution can be a useful tool for novel CIVM characterization, especially with rapidly growing libraries of single-cell data across tissues and developmental time. Overall design: All samples are from a complex in vitro model of neonatal rodent testis. All models are seeded with cells isolated from post-natal day five Sprague Dawleys rats, a complete testis cell mixture was used, without enrichment or depletion of specific cells types. Samples are included for two experiments. The "baseline" experiment, where models seeded from one litter of rats were sampled in technical replicate across 3 days (days 3, 5, and 7). Primary tissue isolates were also sequenced in triplicate for this baseline. The "induction" experiment, where models are maintain in vitro for 10 days, then 50 mIU/mL LH and 20 mIU/mL FSH are added and samples are collected after 2 hours, 6 hours, 12 hours and 48 hours. Samples were also collected on day 10 (time 0) before hormone addition. These are three biological replicates, where three replicates cultures are combined at each timepoint to form one biological replicate (one litter). This is repeated across three litters.