Viability assays
The viability of PCIS were assessed using lactate dehydrogenase (LDH) release (cytotoxicity) assays and water-soluble tetrazolium salt (WST-1) (metabolic) assays (Roche, Mannheim, Germany) from 0-24h in culture. Treatment with the detergent Triton X-100 (MilliporeSigma, Darmstadt, Germany) was included to show maximum LDH release and as a dead-control.
Single cell nuclear sequencing of human colon tissue
Colon tissue samples (n=4), also used to generate PCIS, were snap-frozen and stored at -80oC prior to single nuclear RNA sequencing (scNucSeq). After library generation and quality control, the samples were deep sequenced using a NovaSeq (target 10 000 nuclei, 80 000 reads/nuclei). FASTQ files were aligned to a reference human transcriptome using 10X Genomics Cell Ranger (CA, USA). The resulting gene expression data were merged using the Seurat R package.23,24 Batch effects were removed by “regressing out” variables, and clustering was performed using the Leiden algorithm implemented in Seurat. Cell identities were assigned and confirmed using a list of pre-defined marker genes in reference to single cell gene expression databases and the top distinct genes expressed by each cluster.25,26