Spatiotemporal mosaic patterning of pluripotent stem cells using CRISPR interference

Abstract

Morphogenesis results from the interactions of asymmetric cell populations to form complex multicellular patterns and structures comprised of distinct cell types. However, current methods to model morphogenic events offer little control over parallel cell type co-emergence and do not offer the capability to selectively perturb gene expression in specific subpopulations of cells. We have developed an in vitro system that can spatiotemporally interrogate cell-cell interactions and multicellular organization within human induced pluripotent stem cell (hiPSC) colonies. We examined the effects of independently knocking down molecular regulators of cortical tension and cell-cell adhesion using inducible CRISPRi: Rho-associated kinase-1 (ROCK1) and E-cadherin (CDH1), respectively. Induced mosaic knockdown of ROCK1 or CDH1 in hiPSC populations resulted in differential patterning events within hiPSC colonies indicative of cell-driven population organization. Patterned colonies retained an epithelial phenotype and nuclear expression of pluripotency markers. Gene expression within each of the mixed populations displayed a transient wave of differential expression with induction of knockdown that stabilized in coordination with intrinsic pattern formation. Mosaic patterning of hiPSCs enables the genetic interrogation of emergent multicellular properties of pluripotent cells, leading to a greater mechanistic understanding of the specific molecular pathways regulating the dynamics of symmetry breaking events that transpire during developmental morphogenesis.SIGNIFICANCEHuman embryonic development entails a series of multicellular morphogenic events that lead to primitive tissue formation. Attempts to study human morphogenic processes experimentally have been limited due to divergence from model organisms and the inability of current human in vitro models to accurately control the coincident emergence of heterogeneous cell populations in the spatially controlled manner necessary for proper tissue structure. We developed a human induced pluripotent stem cell (iPSC) in vitro model that enables temporal control over the emergence of heterotypic subpopulations of cells. We examined mosaic knockdown of two target molecules to create predictable and robust cell-patterning events within hiPSC colonies. This method allows for dynamic interrogation of intrinsic cell mechanisms that initiate symmetry breaking events and provides direct insight(s) into tissue developmental principles.

Rho Kinases in Embryonic Development and Stem Cell Research

“…Moreover, studies employing CRISPR gene editing induced specific knockdown of ROCK1 in subpopulations of hiPSC colonies within an otherwise homogeneous population of pluripotent cells. The resulting mosaic knockdown of ROCK1 triggered cellular self-organization within colonies due to the cells lacking ROCK1 moving to the periphery of the colonies while retaining an epithelial pluripotent phenotype, which supports that ROCK1 is a significant player in tissue development and cell organization processes (Libby et al 2018(Libby et al , 2021.…”

Engineering Co-Emergence in Organoid Models

“…Intrinsic organoid engineering approaches would also require a mosaic initial population but differ from the synthetic circuits in one key way: the feedback circuitry should leverage pre-existing interconnected endogenous signaling pathways. This could be accomplished through genome editing in order to direct a subpopulation of PSCs in the initial population to express specific lineage markers (transcription factor editing) [73], modulate patterning events in vitro (secreted factor editing) [65,67], or to achieve a mosaic starting population capable of symmetry breaking and developmental pre-patterning (structural protein editing) [74].…”