Generation of hESC-derived expandable hepatoblast organoids and their derivatives of functional polarized hepatocyte organoids under 3D suspended culture conditions
Background & Aims: The increasing requirements for hepatocytes in therapeutics and pharmacy call for an efficient system that enables large-scale production of functional hepatocytes in vitro. Hepatoblast (HB) hold the ability to regenerate liver when damaged, combined with organoid (org) technology, the culture of human pluripotent stem cell (hPSCs) derived HB-orgs was established under 2D culture system, enabling long-term expansion of HB in vitro. However, the expensive cost and the limitation of existing methods impede the further scale up of the production. Methods: Under 3D suspension culture conditions with the support of Matrigel at low concentration in conjunction with employing the developed (defined) medium, we developed a novel system to efficiently and reproducibly generate expandable HB-orgs and functional polarized hepatocyte organoids (P-hep-orgs) derived from hESCs. HB-orgs were further scaled up by dynamic culture in spinner flasks, and the maturity and function of P-hep-orgs was validated by transcriptome analysis and in vitro and in vivo hepatic functional assays. scRNA-seq was used to further profile HB-orgs and P-hep-orgs. Results: Our HB-orgs exhibited biopotency that could differentiate into functional hepatocytes and cholangiocytes. They proliferated actively at least for 15 passages with defined medium through avoiding the excessive autophagy under 3D suspension culture condition. HB-orgs showed higher proliferation rate in dynamic culture conditions and could reach 10^12 cell number at the initiate number of 3 million cells in only 4 weeks. With further differentiation, P-hep-orgs displayed characteristics of hepatocytes in liver, including the polarization state which mediated by integrin-AMPK signalling pathway, molecular features and improved liver functions. Moreover, P-hep-orgs were used for predicting the toxicity of various compounds, and modeling the development of hepatic lipid accumulation, as well as rescuing acute liver failure of mice. Conclusion: Our work provided a cost-efficient method for large-scale generation of 3D HB-orgs and 3D P-hep-orgs that hold considerable potential to be a robust platform for disease research, drug screening/development and toxic study, as well as clinical applications. Overall design: H9 was cultured on MEFs. MG- HB-orgs were hepatoblasts differentiated from H9 and cultured in our hepatoblast expansion medium without Matrigel supplement. MG+ HB-orgs were hepatoblasts differentiated from H9 and cultured in our hepatoblast expansion medium with Matrigel supplement. NP-hep-spheres were hepatocytes differentiated from our HB-orgs, cultured in HCM without Matrigel supplement. P-hep-orgs were hepatocytes differentiated from our HB-orgs, cultured in HCM with Matrigel supplement. PHH was primary human hepatocytes separated from patient.