Directed differentiation of human pluripotent stem cells into diverse organ-specific mesenchyme of the digestive and respiratory systems.

Development of visceral organs such as the esophagus, lung, liver and stomach are coordinated by reciprocal signaling interactions between the embryonic endoderm and adjacent mesoderm cells in the fetal foregut. While the recent successes in recapitulating developmental signaling <em>in vitro </em>has enabled the differentiation of human pluripotent stem cells (hPSCs) into various types of organ-specific endodermal epithelium, the generation of organ-specific mesenchyme has received much less attention. This is a major limitation in ongoing efforts to engineer complex human tissue. Here, we describe a series of protocols to differentiate hPSCs into different types of organ-specific mesoderm leveraging signaling networks and new molecular markers recently elucidated from single cell transcriptomic analysis of mouse foregut organogenesis. Building on established methods, hPSC-derived lateral plate mesoderm (LPM) treated with either retinoic acid (RA) or RA and a Hedgehog (HH) agonist generates posterior or anterior foregut splanchnic mesoderm respectively (pFG-SpM or aFG-SpM) after 4 days in cultures. These are then directed into organ-specific mesenchyme lineages by the combinatorial activation or inhibition of WNT, BMP, RA or HH pathways from days 4-7 in monolayer culture. By day 7 the cultures are enriched for different types of mesoderm with distinct molecular signatures: 60~90% pure liver septum transversum/mesothelium (STM/Mesothelium), 70~80% pure liver fibroblasts (LF), and populations of ~35% respiratory mesoderm (RM), gastric mesoderm (GM), or esophageal mesoderm (EM). This protocol can be carried out by anyone with moderate experience differentiating hPSCs and provides a novel platform to study human mesoderm development and can be used to tissue engineer more complex foregut organ tissue for disease modeling and regenerative medicine.