Differentiation of human tissues without cilia from pluripotent stem cells

A method of differentiating an isolated genetically modified tubular organoid derived from a genetically modified pluripotent stem cell (PSC), the method comprising:

• (a) providing a quantity of genetically modified PSCs, wherein the genetically modified PSCs comprise a target gene encoding at least one subunit of kinesin-2 and contain a disruption of the target gene that results in the PSCs lacking a ciliary structure;
• (b) culturing the genetically modified PSCs in a first culture medium comprising a ROC kinase inhibitor for at least 24 hours and then culturing the PSC sandwiched between two layers of a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells to form epiblast spheroids, wherein the first medium does not comprise exogenous fibroblast growth factor 2 (FGF2), activin or bone morphogenetic protein;
• and
• (c) contacting the epiblast spheroids from step (b) with a second culture medium comprising at least 12 μM CHIR99021 for at least 24 hours, wherein the second medium does not comprise exogenous fibroblast growth factor 2 (FGF2), activin or bone morphogenetic protein, and
• (d) culturing the epiblast spheroids from step (c) with a third culture medium comprising B27 for at least 48 hours, wherein the third medium does not comprise exogenous fibroblast growth factor 2 (FGF2), activin or bone morphogenetic, thereby differentiating the epiblast spheroids into genetically modified tubular organoids, wherein the genetically modified tubular organoids lack a ciliary structure.

A method for measuring organ specific toxicity and disease phenotypes of an agent on a genetically modified organoid lacking a ciliary structure, the method comprising:

• (a) providing one or more genetically modified organoids lacking a ciliary structure and derived from a genetically modified pluripotent stem cell (PSC) in a high throughput format;
• (b) admixing the agent with the one or more genetically modified organoids;
• and
• (c) detecting one or more outcomes of agent on the one or more genetically modified organoids wherein the one or more outcomes indicates toxicity, disease, differentiation state, or a combination thereof of the one or more genetically modified organoids.