RNA-sequencing with micro-dissected boundary organoid into anterior, posterior, and boundary regions

We report the continuous patterning and dynamic morphogenesis of hepatic, biliary and pancreatic structures, invaginating from a three-dimensional culture of human pluripotent stem cell (PSC). The boundary interactions between anterior and posterior gut spheroids differentiated from human PSCs enables autonomous emergence of hepato-biliary-pancreatic (HBP) organ domains specified at the foregut-midgut boundary organoids in the absence of extrinsic factor supply. The regional identity of the boundary region of anterior-posteior spheroids was analyzed by RNA-sequencing with micro-dissected boundary organoid into anterior, posterior, and boundary regions. Each region expressed the corresponding gut lineage markers. The early HBP specification markers including HHEX1 and PDX1 at the boundary were highly upregulated compared with the other two regions, indicating that the anterior-posterior boundary interactions autonomously generate HHEX and PDX1 positive cells in the absence of exogenous inductive factors. To delineate the HBP progenitor self-inductive mechanism, we evaluated the boundary specific expression profiles of known inductive signaling pathways that includes FGF, BMP, Hedgehog, NOTCH and retinoic acid (RA) signals, and found that the signal downstream of RA were activated prominently at the boundary region but not in the anterior or posterior regions.