Spatiotemporal mosaic self-patterning of pluripotent stem cells using CRISPR interference

Abstract

Morphogenesis involves interactions of asymmetric cell populations to form complex multicellular patterns and structures comprised of distinct cell types. However, current methods to model morphogenic events lack control over cell-type co-emergence and offer little capability to selectively perturb specific cell subpopulations. Our in vitro system interrogates cell-cell interactions and multicellular organization within human induced pluripotent stem cell (hiPSC) colonies. We examined effects of induced mosaic knockdown of molecular regulators of cortical tension (ROCK1) and cell-cell adhesion (CDH1) with CRISPR interference. Mosaic knockdown of ROCK1 or CDH1 resulted in differential patterning within hiPSC colonies due to cellular self-organization, while retaining an epithelial pluripotent phenotype. Knockdown induction stimulates a transient wave of differential gene expression within the mixed populations that stabilized in coordination with observed self-organization. Mosaic patterning enables genetic interrogation of emergent multicellular properties, which can facilitate better understanding of the molecular pathways that regulate symmetry-breaking during morphogenesis.

eLife digest

Embryos begin as a collection of similar cells, which progress in stages to form a huge variety of cell types in particular arrangements. These patterns of cells give rise to the different tissues and organs that make up the body.

Although we often use ‘model’ organisms such as mice and frogs to study how embryos develop, our species has evolved unique ways to control organ development. Investigating these processes is difficult: we cannot experiment on human embryos, and our development is hard to recreate in test tubes. As a result, we do not fully understand how developing human cells specialize and organize.

Libby et al. have now created a new system to study how different genes control cell organization. The system uses human pluripotent stem cells – cells that have the ability to specialize into any type of cell. Some of the stem cells are modified using a technique called inducible CRISPR interference, which makes it possible to reduce the activity of certain genes in these cells.

Libby et al. used this technique to investigate how changes to the activity of two genes – called ROCK1 and CDH1 – affect how a mixed group of stem cells organized themselves. Cells that lacked ROCK1 formed bands near the edges of the group. Cells that lacked CDH1 segregated themselves from other cells, forming ‘islands’ inside the main group. The cells retained their ability to specialize into any type of cell after forming these patterns. However, specific groups of cells were more likely to become certain cell types.

The method developed by Libby et al. can be used to study a range of complex tissue development and cell organization processes. Future work could create human tissue model systems for research into human disease or drug development.