(317g) Modeling liver organogenesis from human stem cells in vitro and in vivo; new approaches in liver tissue engineering
AIChE Annual Meeting
2022
2022 Annual Meeting
Food, Pharmaceutical & Bioengineering Division
Stem Cells and Tissue Engineering
Tuesday, November 15, 2022 - 2:18pm to 2:54pm
During liver organogenesis (LION), E9.5 collectively migrating hepatoblasts (MHs) arise from the E9.0 liver diverticulum (LD) and penetrate the surrounding mesoderm (MES) tissue. The role of the LD in LION has been largely ignored in current human pluripotent stem cell (hPSC) studies. We hypothesize that the LD and surrounding MES form a functional tissue complex (LD-MESC) that triggers LION by simultaneously coordinating exponential growth, collective migration, and differentiation. This can be incorporated into protocols to improve the induction of migrating hepatoblasts.
We first employed systems biology of scRNA-seq data to analyze in vivo LION and support our hypothesis that many biological events are coordinated at the LD-MESC stage. Our analysis of early LION demonstrates that hypoxia, early gene regulatory network (GRN) activation, ALB transcription, exponential growth, collective migration, and metabolic programming are linked. Further, we observe a wide-range of upregulated pathways in signaling (e.g., Hippo), metabolic changes (e.g., oxidative phosphorylation), and migration pathways. To model the LD-MESC concept in vitro, we designed a novel in vivo transplant system, such that the LD could be induced in vivo from definitive endoderm (DE), in the vicinity of mesenchymal cells (MES). Cells were transplanted subcutaneously to model hypoxic growth. For our in vitro studies, we developed a 3-stage protocol with a unique medium formulation. Our system is unique because it employs a single medium throughout, is under completely under hypoxic conditions, and contains no formal instructive factors, thus relying on spontaneous differentiation of gut tube endoderm cells. We performed microscopy and molecular analysis.
The in vivo LD-MESC model demonstrated that subcutaneous transplantation resulted in cord formation similar to hepatic cord morphogenesis, exponential growth, liver gene expression, with no evidence of blood vessels. This is the first reported hPSC system that mimics these important characteristics of early LION. Our 14 day monolayer protocol demonstrated a uniform population of hPSC-derived Hepatoblasts (HBs). Next, we demonstrated successful compaction, and when placed in matrigel droplet culture, we demonstrated collective outward, 3D collective hepatoblast migration for the first time. These cells produced higher ALB, PROX1, and demonstrate Albumin + hepatic cords, and a ~5 fold increase in overall growth. To determine the mechanism by which migration occurs, we evaluated numerous signaling pathways in a chemical screen of growth/migration. W found VT (Hippo) and SU5416 (VEGFR2) were found to inhibit migration, agreeing with the transcriptomic data for murine MH. Interestingly, Hippo pathway is in agreement with our murine MH transcriptomic data, mediates migration and growth of hPSC-MH in vitro. These data substantiates the LD-MESC model, establishes a novel protocol which generates early HBs, and demonstrates a new in vivo model based upon LION principles.
We first employed systems biology of scRNA-seq data to analyze in vivo LION and support our hypothesis that many biological events are coordinated at the LD-MESC stage. Our analysis of early LION demonstrates that hypoxia, early gene regulatory network (GRN) activation, ALB transcription, exponential growth, collective migration, and metabolic programming are linked. Further, we observe a wide-range of upregulated pathways in signaling (e.g., Hippo), metabolic changes (e.g., oxidative phosphorylation), and migration pathways. To model the LD-MESC concept in vitro, we designed a novel in vivo transplant system, such that the LD could be induced in vivo from definitive endoderm (DE), in the vicinity of mesenchymal cells (MES). Cells were transplanted subcutaneously to model hypoxic growth. For our in vitro studies, we developed a 3-stage protocol with a unique medium formulation. Our system is unique because it employs a single medium throughout, is under completely under hypoxic conditions, and contains no formal instructive factors, thus relying on spontaneous differentiation of gut tube endoderm cells. We performed microscopy and molecular analysis.
The in vivo LD-MESC model demonstrated that subcutaneous transplantation resulted in cord formation similar to hepatic cord morphogenesis, exponential growth, liver gene expression, with no evidence of blood vessels. This is the first reported hPSC system that mimics these important characteristics of early LION. Our 14 day monolayer protocol demonstrated a uniform population of hPSC-derived Hepatoblasts (HBs). Next, we demonstrated successful compaction, and when placed in matrigel droplet culture, we demonstrated collective outward, 3D collective hepatoblast migration for the first time. These cells produced higher ALB, PROX1, and demonstrate Albumin + hepatic cords, and a ~5 fold increase in overall growth. To determine the mechanism by which migration occurs, we evaluated numerous signaling pathways in a chemical screen of growth/migration. W found VT (Hippo) and SU5416 (VEGFR2) were found to inhibit migration, agreeing with the transcriptomic data for murine MH. Interestingly, Hippo pathway is in agreement with our murine MH transcriptomic data, mediates migration and growth of hPSC-MH in vitro. These data substantiates the LD-MESC model, establishes a novel protocol which generates early HBs, and demonstrates a new in vivo model based upon LION principles.