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. 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.

背景与研究目的：治疗学与药学领域对肝细胞的需求日益增长，亟需开发一套可在体外大规模生产功能性肝细胞的高效体系。肝母细胞（hepatoblast, HB）在肝脏受损时具备再生能力，结合类器官（organoid, org）技术，科研人员已在二维（2D）培养体系下建立了人多能干细胞（human pluripotent stem cell, hPSCs）来源的肝母细胞类器官（HB-orgs）培养方法，可实现肝母细胞在体外的长期扩增。然而现有方法成本高昂且存在局限，阻碍了该生产流程的进一步规模化。
方法：本研究采用低浓度基质胶（Matrigel）支持的三维（3D）悬浮培养条件，并结合定制的限定性培养基，开发了一套全新的高效且可重复的培养体系，可从人胚胎干细胞（human embryonic stem cell, hESCs）中获取可扩增的肝母细胞类器官，以及功能性极化肝细胞类器官（polarized hepatocyte organoids, P-hep-orgs）。通过转瓶动态培养进一步实现了肝母细胞类器官的规模化扩增，并通过转录组分析、体外及体内肝功能实验验证了极化肝细胞类器官的成熟度与功能。此外，本研究采用单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）对肝母细胞类器官与极化肝细胞类器官进行了转录组图谱解析。
结果：本研究获得的肝母细胞类器官具备分化为功能性肝细胞与胆管细胞的双潜能。在三维悬浮培养条件下，通过限定性培养基避免了过度自噬，该类器官可至少连续传代15次并保持活跃增殖。动态培养条件下的肝母细胞类器官增殖速率更高，仅需4周即可从初始300万细胞扩增至10^12个细胞。经进一步分化得到的极化肝细胞类器官具备肝脏肝细胞的特征，包括由整合素-AMPK信号通路介导的极化状态、分子特征及更优异的肝功能。此外，极化肝细胞类器官可用于预测多种化合物的毒性、构建肝脂质积累疾病模型，以及挽救小鼠急性肝衰竭。
结论：本研究开发了一套低成本的大规模生产三维肝母细胞类器官与三维极化肝细胞类器官的方法，上述类器官具备成为疾病研究、药物筛选/开发、毒性研究及临床应用的可靠平台的巨大潜力。
本研究中，H9细胞在小鼠胚胎成纤维细胞（mouse embryonic fibroblast, MEF）上培养。MG-肝母细胞类器官指由H9细胞分化得到的肝母细胞，在不含基质胶补充的肝母细胞扩增培养基中培养；MG+肝母细胞类器官指由H9细胞分化得到的肝母细胞，在添加基质胶的肝母细胞扩增培养基中培养。NP-肝球指由本研究的肝母细胞类器官分化得到的肝细胞，在不含基质胶补充的肝细胞培养基（hepatocyte culture medium, HCM）中培养；极化肝细胞类器官指由本研究的肝母细胞类器官分化得到的肝细胞，在添加基质胶的肝细胞培养基中培养。PHH指从患者体内分离的原代人肝细胞（primary human hepatocytes）。