(282b) A Miniaturized Organoid Model of Early Liver Development

Liver regenerative medicine is a rich field with many approaches towards organ replacement (liver transplantation) and functional support (bioartificial reactor) approaches. Our approach is to focus on liver development and organogenesis in order to solve problems in liver regenerative medicine. We hypothesize that the structures and cues that initiate three dimensional (3D) liver formation can be mimicked with hepatic organoids. Thus we aimed to engineer an in vitro organoid model of the liver diverticulum (LD), a key miniature structure that: 1) arises in mouse development (E9.5) and human development (d26) and 2) forms the 3D liver. From inside the gut to outside, the LD is composed of a single layer of hepatic endoderm (HE), encased by a single layer of endothelial cells, and surrounded by the septum transversum mesenchyme (STM) containing collagen-rich mesenchyme and a high density of mesenchymal cells. 3D liver formation starts when the hepatic endoderm (HE) delaminates, and together with with endothelial cells, migrates collectively into the STM. We first determined the appropriate LD dimensions with an online mouse database. The LD is an elliptical structure, and we measured a long diameter of 180 µm, a short diameter of 90 µm, and a circumference of 810 µm. The columnar pseudostratified epithelium has an appreciable thickness of 70 µm and is encased in a single layer of endothelial cells. We used HepG2 cells as a model of hepatic endoderm because of their expression of alphafetoprotein (AFP), albumin (Alb), hematopoetically-expressed homeobox (Hex), prospero homeobox 1 (Prox1), and the fact that they represent dedifferentiated hepatocytes. Further, human mesenchymal stem cells (MSC) and human umbilical vein endothelial cells (HUVEC) were used as a model of STM, and LD endothelial cells, respectively. We first lentivirally transduced HepG2 cells with a GFP-Fluc (green flourescent protein and firefly fluciferase) fusion protein which allowed us to quantify hepatic spheroid/organoid viability, proliferation, and assembly. We developed an in vitro model of the LD by first seeding 500 HepG2-GFP-Fluc cells into a round-bottom, low attachment well plate. The HepG2-GFP-Fluc cells self-assembled into a spheroid of 300-400 um in diameter in 72 hours. We performed growth kinetics using daily luminometry on spheroids and demonstrated an 4-fold increase in luciferase activity by day 6 (n =2). To assess HepG2 migratory capacity necessary for liver development, we initially examined 2D migration in a commercially available, modified wound healing assay system. In this system, two square cell islands (500 µm x 500 µm), are separated by 500 µm, and can be tested for migratory potential towards each other. In this case, the leading edge of the HepG2 island moved 50% more distance ( ~175 µm) over 24 hours, when migrating towards an MSC island, compared to a HepG2 island. We further employed a transwell cell invasion assay in which HepG2 cells were plated in the upper chamber and MSC were plated in the lower chamber. HepG2 cells showed a 6-fold increase in migration in the presence of MSCs over the control condition (n =2). To further model the LD, dye-labeled HUVEC were seeded onto the spheroid and imaged for 4 days. HUVEC cells attached to the spheroid surface within 12 hours, and formed 4-5 clusters with 24 hours. By 4 days, these clusters regressed and HUVEC spread across the spheroid, which we now term as organoid. HUVEC-bearing organoids (day 6 of total culture) were submerged in a hydrogel (MG). To analyze organoid growth kinetics, we performed phase microscopy and image analysis. We observed that the mean HUVEC/ HepG2 organoid area increased by 22% over a subsequent 6-day period (n=3). To further assess our spheroid system, we performed qRT-PCR on day 3 spheroid and day 6 organoid cultures. The data suggested maintenance of major HepG2 (and hepatic endoderm) markers Foxa2, HNF4, AFP, Alb, Hex, and Prox1 in both spheroids and organoids compared to HepG2 culture. In summary, we have designed. bioengineered, and characterized a multi-cellular 3D hepatic organoid system which resembles the LD. Future work will assess further aspects of the HUVEC/ HepG2 organoid with respect to liver development, including timing of gene expression, formation of liver like tissue, and function.