Novel lentiviral constructs for the specification of rare and ectopic cell types in human colonoids

Human colonoids derived from adult intestinal tissue provide powerful models for epithelial biology but lack several rare or regionally restricted cell types. To overcome this limitation, we engineered doxycycline-inducible lentiviral constructs to drive expression of transcription factors that control lineage specification, including KLF4, POU2F3, SPIB, NEUROG3, and NEUROG3 T2A NKX6-3. Transduction of human colonoids with these constructs revealed that transient KLF4 induction is sufficient to generate TFF3 and WFDC2 expressing goblet cells. Unexpectedly KLF4 also induced GUCA2A positive colonocytes. In contrast, SPIB and POU2F3 were not sufficient to specify M-cells or mature tuft cells respectively, suggesting the requirement for additional permissive cues. Notably, co-expression of NEUROG3 and NKX6-3 induced enteroendocrine subtypes characteristic of the duodenum. These findings establish a set of lentiviral tools for induction of specific epithelial lineages in human colonoids providing a platform for replenishing cell types that are lost or altered in intestinal diseases.