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Single-cell analysis of population context advances RNAi screening at multiple levels.

ABSTRACT: Isogenic cells in culture show strong variability, which arises from dynamic adaptations to the microenvironment of individual cells. Here we study the influence of the cell population context, which determines a single cell's microenvironment, in image-based RNAi screens. We developed a comprehensive computational approach that employs Bayesian and multivariate methods at the single-cell level. We applied these methods to 45 RNA interference screens of various sizes, including 7 druggable genome and 2 genome-wide screens, analysing 17 different mammalian virus infections and four related cell physiological processes. Analysing cell-based screens at this depth reveals widespread RNAi-induced changes in the population context of individual cells leading to indirect RNAi effects, as well as perturbations of cell-to-cell variability regulators. We find that accounting for indirect effects improves the consistency between siRNAs targeted against the same gene, and between replicate RNAi screens performed in different cell lines, in different labs, and with different siRNA libraries. In an era where large-scale RNAi screens are increasingly performed to reach a systems-level understanding of cellular processes, we show that this is often improved by analyses that account for and incorporate the single-cell microenvironment.

SUBMITTER: Snijder B

PROVIDER: S-EPMC3361004 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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Single-cell analysis of population context advances RNAi screening at multiple levels.

Snijder Berend B Sacher Raphael R Rämö Pauli P Liberali Prisca P Mench Karin K Wolfrum Nina N Burleigh Laura L Scott Cameron C CC Verheije Monique H MH Mercer Jason J Moese Stefan S Heger Thomas T Theusner Kristina K Jurgeit Andreas A Lamparter David D Balistreri Giuseppe G Schelhaas Mario M De Haan Cornelis A M CA Marjomäki Varpu V Hyypiä Timo T Rottier Peter J M PJ Sodeik Beate B Marsh Mark M Gruenberg Jean J Amara Ali A Greber Urs U Helenius Ari A Pelkmans Lucas L

Molecular systems biology 20120424

Isogenic cells in culture show strong variability, which arises from dynamic adaptations to the microenvironment of individual cells. Here we study the influence of the cell population context, which determines a single cell's microenvironment, in image-based RNAi screens. We developed a comprehensive computational approach that employs Bayesian and multivariate methods at the single-cell level. We applied these methods to 45 RNA interference screens of various sizes, including 7 druggable genom ...[more]

PMID: 22531119

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Project description:The dental pulp plays a crucial role in the long-term maintenance of tooth function. The progress of endodontic treatment and pulp tissue regeneration engineering has made pulp-regeneration therapy promising in clinical practice. However, the mechanisms of pulp regeneration and the role of dental stem cells in development and regeneration have not been fully elucidated. Bridging the gaps between clinical operation and basic research is urgently needed. With the application of single-cell sequencing technology in dental research, the landscapes of human dental pulp cells have begun being outlined. However, the specific cellular heterogeneity of dental pulp cells, especially that of dental stem cells, at different spatial and temporal levels, is still unclear. In this study, we used single-cell RNA sequencing analysis of pulp samples at four different developmental stages and combined the findings with immunohistochemical staining to explore the development of dental pulp and stem cells. The results revealed temporal changes in the proportion of pulp cells during development. For example, mononuclear phagocytes accounted for a higher proportion in early samples. Odontoblasts identified by DMP1 had a higher expression of ion channel-related and neurodevelopment-related genes. Subpopulations were identified in fibroblasts, odontoblasts, and mesenchymal stem cells. We identified a subclass of odontoblasts that expresses DGKI and RRBP1 present in early developmental samples. A population of earlier mesenchymal stem cells expressed the SEPTIN gene, which may have greater proliferative and differentiation potential. Furthermore, dental pulp stem cells can differentiate into two directions: mineralization and myogenesis. In summary, the specific cellular heterogeneity of dental pulp cells was revealed at different spatial and temporal levels. These findings may shed light on the mechanism of tooth development. The gene expression profile of developing pulp cells may help to select cells for regenerative engineering and improve the success of dental pulp regeneration.

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Single-cell analysis of population context advances RNAi screening at multiple levels.

Publications

Single-cell analysis of population context advances RNAi screening at multiple levels.

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