RT Journal Article
SR Electronic
T1 Dynamic changes in the niche and transcription trigger early murine and human pluripotent stem cell-derived liver organogenesis
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2022.07.24.501313
DO 10.1101/2022.07.24.501313
A1 Ogechi Ogoke
A1 Daniel Guiggey
A1 Sarah Thompson
A1 Alexander Chiang
A1 Tram Hoang Anh Nguyen
A1 Daniel Berke
A1 Cortney Ott
A1 Allison Kalinousky
A1 Claire Shamul
A1 Peter Chen
A1 Shatoni Ross
A1 Zhaowei Chen
A1 Pooja Srivastava
A1 Supriya Mahajan
A1 Ruogang Zhao
A1 Rudiyanto Gunawan
A1 Natesh Parashurama
YR 2022
UL http://biorxiv.org/content/early/2022/08/10/2022.07.24.501313.abstract
AB Organoids bearing human stem cell-derived progenitors enable basic and applied investigation of organogenesis in a wide range of epithelial tissues. During liver organogenesis (LO), E9.5 collectively migrating hepatoblasts (MHs) arise from the E9.0 liver diverticulum (LD) and directly penetrate the surrounding mesoderm (MES) tissue, forming cell strands that link migration, differentiation, and growth. Currently, human pluripotent stem cell (hPSC) organoid protocols model the E10.5 liver bud and forward differentiation, but not the LD or the LD-derived MHs, in spite of their significance. In fact, the transcriptome underlying MHs, the niche that drives their migration, and methods to induce them from hPSC remain key questions.We performed bioinformatics analysis of single cell RNA-seq data, in vivo transplantation, and in vitro hPSC differentiation with organoid formation, microscopy, gene and protein expression, small molecule inhibitor screening of growth, and organoid culture in bioengineered devices to assess tissue tension.Our in depth bioinformatic analysis of early murine LO demonstrates pathway up-regulation of an unexpected wide array of soluble signaling factors, as well as cell cycle, chromatin modification, and metabolic reprogramming, in addition to a widespread cell stress-response. These findings led us hypothesize that the LD and MES tissue form a tissue complex (LD-MESC) that drives MH induction. Using this LD-MESC concept, we designed an in vivo transplant system, as well as a three-step in vitro protocol for inducing hPSC-derived MHs, both of which recapitulate liver growth, morphogenesis, differentiation. We show that Hippo signaling pathway, in agreement with murine MH data, mediates migration and growth of hPSC-MH in vitro. These data substantiate the LD-MESC model developed here, and directly address key challenges facing liver regenerative medicine.Our bioinformatics, in vitro, and in vivo data all support the concept that the LD-MESC initiates LO. This concept can be used to change protocols to emphasize linking of migration, growth, with differentiation. Modeling epithelial collective migration for LO bolsters not only organogenesis studies of alternate endodermal organs, but also in vivo transplantation efforts, and facilitates employing migrating organoids to therapeutically target human tumor migration/metastasis.Competing Interest StatementThe authors have declared no competing interest.AFPalpha-fetoproteinALBalbuminBMP4Bone Morphogenetic ProteinBSABovine serum albuminCCMCollective cell migrationCHIRWnt pathway agonistDMEMDulbecco’s Modified Eagle’s MediumEDTAEthylenediaminetetraacetic acidEGFEpidermal growth factorEGM-2Endothelial growth medium 2EHTepithelial to hepatic transitionFBSfetal bovine serumFGF2Fibroblast growth factor-2iPSCInduced pluripotent stem cellsHEPHepatocyteH + Mhepatic and mesenchymalhESChuman embryonic stem cellshPSChuman pluripotent stem cellsHBshepatoblastsHSCshematopoietic stem cellsHEhepatic endodermHFFhuman foreskin fibroblastsHGFhepatocyte growth factorsHPSCHuman pluripotent stem cellsIMDMIscove’s modified Dulbecco’s mediumLDliver diverticulumMESMesodermMGMatrigel (growth-factor free)PBSTPhosphate buffered saline tween 20RT-PCRReal-time polymerase chain reactionR3 IGF-1R3-Insulin growth factor-1SFDSerum free-differentiationSTMSeptum transversum mesenchymeTFsTranscription factorsVEGFVascular endothelial growth factor