When I was a kid, I used to read a lot of comic books. The idea of super powers fascinated me (am I alone in this, as a biologist?). Amongst these comics was one called New Mutants, which was kind of like a teenage version of X-Men.
New Mutants is also a good way to describe our new paper. It describes a new class of stem cells that doesn't normally exist in nature, and has a few superpowers. The secret to these stem cells is that they lack a tiny appendage, the primary cilium, which is a microtubule-based organelle located at the cell surface. This structure - like an antenna - helps organize the cell and control its behaviors. Without it, the cells are a bit like Magneto without his helmet. They can't control themselves very well, and go off in mysterious directions.
In nature, dysfunction of the cilium causes a class of genetic disorders called ciliopathies. These are often syndromic and cause a spectrum of clinical manifestations affecting various organs, including the brain, kidneys, retina, and liver. This group of disorders is genetically heterogeneous, associated with many genes. But what all of these appear to have in common is a link to the primary cilium. Primary cilia are present in most cell types and organ systems and are associated with key signaling pathways that orchestrate cell polarity, proliferation, development, organogenesis, and tissue homeostasis. Cilia are also important for certain types of cancer, such as medulloblastoma. To create a general tool for studying cilia in humans, we sought to disrupt these tiny organelles in human pluripotent stem cells, hoping that this might illuminate disease processes.
When we started this project, we weren't even sure it would be possible to make these kinds of stem cells. From our previous work, we knew that pluripotent stem cells were ciliated, so we took a gamble that the cilia weren't critical for these stem cells to survive. To test this, we used CRISPR-Cas9 to knock out KIF3A, a motor protein required for ciliogenesis in other systems. The gamble paid off - the KIF3A mutants lost their cilia, but appeared to be healthy and normal pluripotent stem cells! Importantly, disruption of cilia did not affect stem cell self-renewal, polarity, and chromosomal stability. This meant that we could use the cells for further experiments - and since the cells were immortal, they could become a permanent resource for the research community.
To really build out this resource, and make sure the effects we observed were generalizable, we went ahead and knocked out another gene, KIF3B. And we made these mutants in two genetic backgrounds: in the human embryonic stem (ES) cell line WA09 (WiCell, female), and the induced pluripotent stem (iPS) cell line WTC11 (Gladstone Institute, male). Ultimately, we generated 7 KIF3A-/- mutants, 7 KIF3B-/- mutants and 12 isogenic controls, making this a very large and comprehensive set of mutants. Immunostaining confirmed that KIF3A-/- and KIF3B-/-stem cells lack primary cilia.
To investigate ciliopathy-associated phenotypes, we proceeded to generated kidney organoids lacking primary cilia. When subjected to our standard differentiation protocol, we observed a drastic reduction of kidney organoids arising from mutant stem cells, compared to isogenic controls. The organoid differentiations were consistently impaired in all the mutants, and over several attempts. We could see the organoids beginning to form in these cultures in large numbers, but then these appeared to melt away, leaving only a few structures for us to study.
At first, this was rather frustrating to us, and seemed a bottleneck to our goal of studying cilia-associated phenotypes, such as polycystic kidney disease. But soon it became evident that this phenotype was specific for loss of cilia. This led to our first 'Aha' moment: Cilia were important for making organoids! Stem cells could make simple cell types just fine without cilia, but they struggled to make architecturally complex structures, including not only kidney organoids, but also neuronal organoids, as well as bona fide human tissues such as cartilage. We traced this to a defect in hedgehog signaling, a pathway that 'operates' through primary cilia. Without cilia, hedgehog signaling wasn't being controlled properly (think Magneto here), resulting in a cellular identity crisis. Although gene expression networks in kidney organoids have been extensively studied, this was the first time hedgehog signaling was shown to be important for their formation.
What about superpowers? One of the most interesting features of ciliopathy disorders is the tendency of kidney tubules to grow into expansive cysts. We isolated the few mutant kidney structures that arose from the differentiations and subjected them to the cystogenesis assay we previously developed for organoids with polycystic kidney disease mutations. It was exciting to see that the tubules of mutant organoids expanded into translucent fluid-filled cysts that were specific to the mutants. Over several months, these organoids grew into very large structures - some of our favorites were about the size of a golf ball! These had expanded a million-fold or so in size. We had a lot of fun taking photo shoots and movies of these organoids using our cell phones, and some of those data ended up in the paper! At a scientific level, these results revealed that kidney organoids can recapitulate ciliopathy-associated renal phenotypes at the tissue scale.
Ultimately, our early struggles trying to make organoids from these cilia knockout cells illustrated one of the main advantages of the stem cell system. Differentiation of organoids mimics development in vivo and distinct stages are recapitulated. This allowed us to assess the early role of cilia in controlling hedgehog during kidney organogenesis, as well as the later role of cilia in inhibiting cystic growth after differentiation. It's easier to dissect out these stages in stem cells, compared to an animal model, in which every step depends so critically on the one before. Our lab had never studied hedgehog before, thus these cilia knockout cells took us on quite a journey. There's quite a lot left to learn from them as they continue to discover their superpowers. (What do you think would be interesting? Let us know in the comments!)
In closing, I want to give a special thanks to our entire team, especially Nelly, who created these super-powered cells, parented them, and helped me write this blog post; our intrepid funders and supportive academic environment; and our editor, who was gracious and patient in helping us get this important story told. 'Nuff said!