Abstract
Three-dimensional (3D) collective cell migration (CCM) is critical for improving liver cell therapies, eliciting mechanisms of liver disease, and modeling human liver development and organogenesis. Mechanisms of CCM differ in 2D vs. 3D systems, and existing models are limited to 2D or transwell-based systems, suggesting there is a need for improved 3D models of CCM. To recreate liver 3D CCM, we engineered in vitro 3D models based upon a morphogenetic transition that occurs during liver organogenesis, which occurs rapidly between E8.5 and E9.5 (mouse). During this morphogenetic transition, 3D CCM exhibits co-migration (multiple cell types), thick-strand interactions with surrounding septum transversum mesenchyme (STM), branching morphogenesis, and 3D interstitial migration. Here, we engineer several 3D in vitro culture systems, each of which mimics one of these processes in vitro. In mixed spheroids bearing both liver cells and uniquely MRC-5 (fetal lung) fibroblasts, we observed evidence of co-migration, and a significant increase in length and number of liver spheroid protrusions, which was highly sensitive to transforming growth factor beta 1 (TGFβ1) stimulation. In MRC-5-conditioned medium (M-CM) experiments, we observed dose-dependent branching morphogenesis associated with an upregulation of Twist1, which was inhibited by a broad TGFβ inhibitor. In models in which liver spheroids and MRC-5 spheroids were co-cultured, we observed complex strand morphogenesis, whereby thin, linear, 3D liver cell strands attach to the MRC-5 spheroid, anchor and thicken to form permanent and thick anchoring contacts between the two spheroids. In these spheroid co-culture models, we also observed spheroid fusion and strong evidence for interstitial migration. In conclusion, we present several novel cultivation systems that recreate distinct features of liver 3D CCM. These methodologies will greatly improve our molecular, cellular, and tissue-scale understanding of liver organogenesis, liver diseases like cancer, and liver cell therapy, and will also serve as a tool to bridge conventional 2D studies and preclinical in vivo studies.
Highlights
Liver cell migration plays important roles in liver organogenesis, disease, and therapy
Gene expression analysis of 3D spheroids, compared with controls, demonstrates a decrease in Foxa2 transcriptional expression, but no significant changes in AFP, albumin, and TTR (Figure 1G), and histological analysis demonstrates no central necrosis by day 3 (Figure 1H). Using this 3D culture, we modeled the septum transversum mesenchyme (STM) that is present in the liver diverticulum (LD) with 20,000 human mesenchymal stem cells (hMSC) within MG (Figure 1I)
While we expected the liver cells to migrate toward the MSC, we instead observed that the hMSC migrated toward the liver spheroids within 24h and over a 3-day period (Figure 1J and Supplementary Figure 3A) and the data demonstrated no changes in circularity and decreasing distance from hMSC and liver spheroid (Figure 1K)
Summary
Liver (epithelial) cell migration plays important roles in liver organogenesis, disease, and therapy. Studies of rat fetal hepatoblast expression indicate that genes associated with 3D CCM, morphogenesis, and extracellular matrix remodeling, are highly upregulated in fetal hepatoblasts (Petkov et al, 2000). This strongly suggests that interstitial migration plays a role in fetal liver growth. Liver 3D CCM is important for successful liver cell therapy, in which either adult or fetal hepatocytes are employed for acute and chronic liver disease models. 3D liver CCM is important for liver development, interstitial migration during fetal liver growth, HCC, and liver cell therapy
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