Differentiation of Human Intestinal Organoids with Endogenous Vascular Endothelial Cells

Abstract

Human pluripotent stem cell (hPSC)-derived intestinal organoids (HIOs) generated using directed differentiation lack some cellular populations found in the native organ, including vasculature. Using single cell RNA sequencing (scRNAseq), we have identified a transient population of endothelial cells (ECs) present early in HIO differentiation that are lost over time in culture. Here, we have developed a method to enhance co-differentiation and maintenance of ECs within HIOs (vHIOs). Given that ECs are known to possess organ specific gene expression, morphology and function, we used bulk RNAseq and scRNAseq to interrogate the developing human intestine, lung, and kidney in order to identify organ-enriched EC-gene signatures in these organ systems. By comparing organ-specific gene signatures along with markers validated by fluorescent in situ hybridization to HIO ECs, we find that HIO ECs grown in vitro share the highest similarity with native intestinal ECs relative to kidney and lung. Together, these data show that HIOs can co-differentiate a native EC population that are properly patterned with an intestine-specific EC transcriptional signature in vitro..

Induced organoids derived from patients with ulcerative colitis recapitulate colitic reactivity

“…5a ). The presence of neural cells has been recently reported in human intestinal organoids 75 . Here we show a significant increase in these cell classes, as well as immune and stromal cells in the UC compared to the normal organoids (Supplementary Fig.…”

Intestinal Morphogenesis in Development, Regeneration, and Disease: The Potential Utility of Intestinal Organoids for Studying Compartmentalization of the Crypt-Villus Structure

“…As cell-to-cell interactions with stromal cells including mesenchymal fibroblast are important in crypt/villus morphogenesis (Walton et al, 2016), it is necessary to develop organoid culture method with various cell composition. Recently, as intestinal organoid differentiation methods composed of multi-lineage cells or technologies for co-culture with various cells are actively developed, these limitations are expected to be overcome in the near future (Workman et al, 2017;Holloway et al, 2020). Second, it is necessary to develop organoid culture methods that enable changes to the environmental factors according to the crypt/villus developmental process.…”

Chromatin regulatory dynamics of early human small intestinal development using a directed differentiation model

“…To determine whether the differences in HOX gene expression between early Duo and Ile extends beyond this in vitro directed differentiation model, we mined the single cell RNA-seq (scRNA-seq) dataset of primary human Duo and Ile tissue at very early stages of fetal development ( 13 , 43 ). Specifically, we focused on matched Duo and Ile fetal tissue at week 11, 14 and 19 of gestation.…”

“…The scRNA-seq data of primary human fetal tissues was generated in recent studies ( 13 , 43 ). Briefly, tissue samples were processed into single cell suspension detailed in ( 13 , 43 ).…”

“…The scRNA-seq data of primary human fetal tissues was generated in recent studies ( 13 , 43 ). Briefly, tissue samples were processed into single cell suspension detailed in ( 13 , 43 ). As described in ( 13 ), the scRNA-seq dataset of human developing gastrointestinal organs and multiple gestational time points were generated by 10× Genomics platform and mapped to hg19 using default alignment parameters provided by Cell Ranger.…”