Organoids Derived from Cilia-Free Stem Cells Model Ciliopathy Phenotypes

Almost every eukaryotic cell type has cilia—tiny hairlike structures—on their surface. But, the function of cilia is not well understood. Now, researchers show that human pluripotent stem cells (hPSCs) in which the kinesin-2 subunits KIF3A or KIF3B have been knocked out, can be used to reveal ciliary functions. The cells, genetically edited using CRISPR, also model ciliopathy phenotypes. In addition, human tissues and organoids derived from these cilia-free stem cells manifested ciliopathy-like symptoms.

This work is published in Nature Biomedical Engineering in the article, “Modeling ciliopathy phenotypes in human tissues derived from pluripotent stem cells with genetically ablated cilia.”

“We are trying to understand what cilia do, so we ablated them from these cells,” said Benjamin Freedman, PhD, associate professor of medicine at the University of Washington School of Medicine. “We wanted to see if the cells would re-create symptoms of ciliopathy without the cilia. Sure enough, when we turned the cells into tissues and organoids (tissue-like structures), they re-created polycystic kidney disease and problems with brain development.”

The cilia-knockout stem cells “represent a powerful new tool for understanding this group of diseases, which can be used to guide therapy development,” said Freedman.

The KIF3A^–/– and KIF3B^–/– hPSCs remained robustly self-renewing and pluripotent while lacking cilia. Tissues and organoids, the authors noted, derived from these hPSCs displayed phenotypes that recapitulated defective neurogenesis and nephrogenesis, polycystic kidney disease, and other features of the ciliopathy spectrum.

There are at least 15 ciliopathies—diseases that emerge from defects in cilia—each rare in terms of population prevalence and each with its own constellation of partially overlapping symptoms. Ciliopathies frequently present at birth; an exception is polycystic kidney disease, which affects about 1 in 500 people and generally causes clinical problems later in life.

Because ciliopathies affect many organs, pluripotent stem cells could offer a “one-stop shop” to study these diseases.

In removing cilia from human pluripotent stem cells, Freedman and his colleagues sought to understand what would happen in their subsequent transformation into tissues and organoids. As it happened, the cilia-free stem cells appeared normal but were unable to fully realize new forms.

“It was surprising to me that, at a certain point after they were turning into tissues, they seemed to break down,” Freedman said. “They struggled to transform into anything sophisticated. I think one lesson from this is that the cilia help get cells through their final stage of development.”

The authors also found that “human cilia mediate a critical switch in hedgehog signaling during organoid differentiation, and they constitutively release extracellular vesicles containing signaling molecules associated with ciliopathy phenotypes.”

It was first reported in 2000 that polycystic kidney disease could stem from defects in cilia, but the mechanism of damage that causes cysts to form has escaped scientists. By creating cilia-free stem cells that harbor disease, Freedman said, the researchers now have a framework with which to test and compare molecular actions in the cilia.

“By comparing cells that totally lack cilia to cells that possess cilia but lack PKD genes, as well as to normal cells, we have the whole range of cell types that should enable us to deduce what’s going on among the molecules involved. For almost 30 years we’ve known the genes involved in PKD—even before we knew that cilia were implicated. Hopefully having these distinct cell types will enable us to figure out what specific disruption these genetic molecules are causing to create PKD.”