Mesenchyme modulates three-dimensional, collective cell migration of liver cells in vitro - a role for TGFβ pathway

Three dimensional (3D) collective cell migration (CCM) is critical for improving liver cell therapies, eliciting mechanisms of liver disease, and modeling human liver development/ organogenesis. Here, we modeled liver organogenesis to induce 3D CCM and improve existing models. The liver diverticulum, normally surrounded by septum transversum mesenchyme (STM) at E8.5, was modeled with a miniature liver spheroid surrounded by mesenchymal cells and matrix. In mixed spheroid models with both liver and uniquely MRC5 (fetal lung) fibroblasts, we observed co-migration of cells, and a significant increase in length and number of liver spheroid protrusions, and this was highly sensitive to TGFB1 stimulation. To understand paracrine effects between MRC-5 cells and liver, we performed conditioned medium (M-CM) experiments. Interestingly, the addition of M-CM increased liver 3D CCM, with thin, 3D, dose-dependent branching morphogenesis, an upregulation of Twist1, and a sensitivity to a broad TGFB inhibitor. To test the effects of cell-cell interactions of 3D CCM, the STM was modeled with a spheroid of MRC-5 cells, and we performed co-spheroid culture of liver with MRC-5. We observed a complex 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. We also observed spheroid fusion, a form of interstitial migration. In conclusion, we present several novel cultivation systems that induce distinct features of 3D CCM, as judged by the presence of branching, linearity, thickness, and interstitial migration. These methodologies will greatly improve our molecular, cellular, and tissue-scale understanding of liver organogenesis, liver diseases, and liver cell therapy, and will serve as a tool to bridge conventional 2D studies and preclinical in vivo studies.