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

In the past 15 years, organoid models have revolutionized the study of the human gastrointestinal (GI) epithelium. Organoids can be derived from patient tissue and grown as strictly epithelial structures referred to as enteroids and colonoids. Despite their utility, rare cell types are often absent in enteroids and colonoids. We hypothesized that induced expression of key transcription factors (TFs) would be sufficient to specify a subset of these cell types. To test this hypothesis, we generated lentiviral constructs that allow the inducible expression of cell-type specifying TFs. We transduced human enteroids and colonoids with these constructs and examined their ability to induce differentiated cell types. Overall design: To determine the sufficiency of TFs for specifying cell types in enteroids and colonoids, cDNAs for POU2F3 which is required for tuft cell differentiation, SPIB which is required for M-cell differentiation, and KLF4 which is required for goblet cell differentiation, were individually cloned into a doxycycline inducible lentiviral vector. A lentiviral NEUROG3 construct has been previously shown to induce enteroendocrine cells in enteroids and human pluripotent stem cell derived gastrointestinal organoids. NEUROG3 is required for enteroendocrine cell specification in the GI tract. To determine if ectopic duodenal enteroendocrine cells could be generated in colonoids, we generated a NEUROG3-T2A-NKX6-3 construct. NKX6-3 is required for generation of gastrin-producing G cells and somatostatin-producing D cells. The resultant constructs were packaged into lentiviral particles which were used to transduce human enteroids/colonoids