Differences between in vivo and ex vivo hematopoietic model systems modulate the outcomes of genetic perturbations.

Ex vivo cell cultures are reductionist models that enable cost effective, precisely controlled experiments with fewer ethical concerns than in vivo conditions. This results in their extensive use in diverse studies, especially large-scale genetic and drug perturbation screens. Although it is commonly accepted that ex vivo models do not fully recapitulate in vivo conditions, the molecular effects of model systems on cells in homeostasis and on cells undergoing drug or genetic perturbations are poorly understood. Using a CRISPR knockout (KO) screen with transcriptome read-out (Perturb-seq) of hematopoietic progenitor cells cultured ex vivo and grown in vivo, we analyzed the effects of ex vivo culture on unperturbed and perturbed cells. Unperturbed cells cultured ex vivo generally showed reduced basal interferon signatures and increased growth and metabolism signatures. These differences in unperturbed cells translated to differences between KO effects observed in vivo and ex vivo. We validated this impact of the model system on KO effects in an additional dataset of genetic KOs in bulk-sorted splenic immune cells, which confirmed our results. Interestingly, genes and molecular pathways with different KO effects were partly predicted by differences between unperturbed cells cultured in vivo and ex vivo. We therefore evaluated the performance of state-of-the-art models in predicting in vivo KO effects from ex vivo KO effects. This proved challenging, demonstrating the need for further developments. In summary, our study reveals differences in culture models, suggests approaches to improve culture models, and provides a test case for computational predictions of perturbation effects.