Development of functional resident macrophages in human pluripotent stem cell-derived colonic organoids and human fetal colon [bulk rna-seq]

Most organs have tissue resident immune cells that function during tissue development, homeostasis and disease. Human organoids, derived either from adult tissues or from pluripotent stem cells (PSCs), have been lacking these immune cells, which limits their utility to model many normal and disease processes. Here we describe that human PSC-derived colonic organoids (HCOs) co-develop a diverse population of immune cells. Comparisons to the developing human hematopoietic cells, demonstrate that HCO cultures developed hemogenic endothelium and erythromyeloid progenitors that undergo stereotypical steps in differentiation, resulting in the generation of macrophages. HCO macrophages acquired a transcriptional signature most similar to human fetal small and large intestine tissue-resident macrophages. Functional analyses demonstrate that HCO-macrophages modulate cytokine secretion in response to pro- and anti-inflammatory signals. In addition, macrophages were able to phagocytose and mount a robust response to pathogenic bacteria. In HCOs that were transplanted into mice, macrophages expanded within the colonic organoid tissue, established a close association with the colonic epithelium and were not displaced by the host bone marrow-derived macrophages. These studies support the conclusion that hemogenic endothelium in HCOs gives rise to multipotent hematopoietic progenitors and functional tissue-resident macrophages. Human pluripotent stem cell derived intestinal and colonic organoids were grown in vitro for 21 and 35 days and total RNA was extracted from pooled organoids and subjected to bulk 3' RNA-sequencing. All conditions were done in biological triplicate. Human pluripotent stem cell derived intestinal and colonic organoids were generated using modified clumps (mod) or human colonic organoids using standard grown 35 days and total RNA was extracted from pooled organoids and subjected to bulk 3' RNA-sequencing. All conditions were done in biological triplicate except HIO (mod) was performed in biological duplicate.