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Generation of esophageal organoids from human pluripotent stem cells and their use to study human development

ABSTRACT: Tracheoesophageal disorders and diseases are prevalent in humans such that an organoid model of human esophagus could be greatly beneficial. We therefore established a three-dimensional esophageal organoid culture system through the directed differentiation of human pluripotent stem cells (hPSCs). We identified that sequential manipulation of several signaling pathways resulted in patterning of definitive endoderm to dorsal anterior foregut spheroids (dAFGs). Outgrowth of dAFGs for 1-2 months resulted in human esophageal organoids (HEOs) with a stratified squamous epithelium comparable to a late gestation mouse embryonic esophagus. These 1 and 2 month old HEOs were harvested for RNA to transcriptionally profile and compare them to profiles of esophageal tissue biopsies and keratinocytes. We then used HEOs and mouse embryos to identify how SOX2 mediates separation of the esophageal and respiratory lineages and found that loss of endodermal Sox2 results in complete esophageal agenesis. Using a SOX2 CRISPR interference iPS line, we generated dorsal and ventral anterior foregut progenitors (by manipulating BMP signaling) with or without SOX2-knockdown. At the transcriptional level, SOX2 acts to promote esophageal specification in both mice and humans in part by inhibiting Wnt signaling in the dorsal AFG and promoting survival of esophageal epithelium. In addition to this use of hPSC-derived esophageal organoids to study development, HEOs can be used for future studies of esophageal pathologies, such as Barrett’s metaplasia and carcinoma.

ORGANISM(S): Homo sapiens

PROVIDER: GSE112886 | GEO | 2018/07/01

REPOSITORIES: GEO

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Project description:Underdeveloped lungs are the primary cause of death in premature infants, however, little is known about stem and progenitor cell maintenance during human lung development. In this study, we have identified that FGF7, Retinoic Acid and CHIR-99021, a small molecule that inhibits GSK3 to activate Wnt signaling, support in vitro maintenance of primary human fetal lung bud tip progenitor cells in a progenitor state. Furthermore, these factors are sufficient to derive a population of human bud tip-like progenitor cells in 3D organoid structures from human pluripotent stem cells (hPSC). Functional studies showed that hPSC-derived bud tip progenitor organoids do not contain any mesenchymal cell types, maintain multilineage potential in vitro and are able to engraft into the airways of injured mice and respond to systemic factors. We performed RNA-sequencing to assess the degree of similarity in global gene expression profiles between the full human fetal lung (59-127 days gestation), isolated human fetal bud tip progenitors, organoids grown from primary fetal bud tip progenitors, and hPSC-derived bud tip organoids. Results showed that hPSC-derived organoids have molecular profiles similar to organoids generated from primary human fetal lung tissue. Gene expression differences between hPSC-derived bud tip organoids and fetal progenitor organoids may be related to the presence of contaminating mesenchymal cells in primary cultures. hPSC-derived bud tip organoids are generated from a well-defined human cell sources, offering a distinct advantage over rare primary tissue as a means to study human specific lung development, homeostasis and disease. Sample Nomenclature - Description ------------------------------------------------------------------------- Peripheral fetal lung the distal/peripheral portion of the fetal lung (i.e., distal 0.5 cm) was excised from the rest of the lung using a scalpel. This includes all components of the lung (e.g., epithelial, mesenchymal, vascular). Isolated fetal bud tip the bud peripheral portion of the fetal lung was excised with a scalpel and subjected to enzymatic digestion and microdissection. The epithelium was dissected and separated from the mesenchyme, but a small amount of associated mesenchyme likely remained. Fetal progenitor organoid 3D organoid structures that arose from culturing isolated fetal epithelial bud tips. Foregut spheroid 3D foregut endoderm structure as described in Dye et al. (2015). Gives rise to patterned lung organoid (PLO) when grown in 3F medium. Patterned lung organoid (PLO) lung organoids that were generated by differentiating hPSCs, as described throughout the manuscript. Bud tip organoid organoids derived from PLOs, enriched for SOX2/SOX9 co-expressing cells, and grown/passaged in 3F medium.

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Generation of esophageal organoids from human pluripotent stem cells and their use to study human development

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