Lekkala Vinod Kumar Reddy, Shrestha Sunil, Qaryoute Ayah Al, Dhinoja Sanchi, Acharya Prabha, Raheem Abida, Jagadeeswaran Pudur, Lee Moo-Yeal
Department of Biomedical Engineering, University of North Texas, Denton, Texas, USA.
Department of Biological Sciences, University of North Texas, Denton, TX, USA.
bioRxiv. 2024 Aug 22:2024.08.21.608997. doi: 10.1101/2024.08.21.608997.
Liver tissues, composed of hepatocytes, cholangiocytes, stellate cells, Kupffer cells, and sinusoidal endothelial cells, are differentiated from endodermal and mesodermal germ layers. By mimicking the developmental process of the liver, various differentiation protocols have been published to generate human liver organoids (HLOs) using induced pluripotent stem cells (iPSCs). However, HLOs derived solely from the endodermal germ layer often encounter technical hurdles, such as insufficient maturity and functionality, limiting their utility for disease modeling and hepatotoxicity assays. To overcome this, we separately differentiated EpCAM endodermal progenitor cells (EPCs) and mesoderm-derived vascular progenitor cells (VPCs) from the same human iPSC line. These cells were then mixed in BME-2 matrix and concurrently differentiated into vascular human liver organoids (vHLOs). Remarkably, vHLOs exhibited significantly higher maturity than vasculature-free HLOs, as demonstrated by increased coagulation factor secretion, albumin secretion, drug-metabolizing enzyme (DME) expression, and bile acid transportation. To enhance assay throughput and miniaturize vHLO culture, we 3D bioprinted expandable HLOs (eHLOs) in BME-2 matrix on a pillar plate platform derived from EPCs and VPCs and compared with HLOs derived from endoderm alone. Compared to HLOs cultured in a 50 μL BME-2 matrix dome in a 24-well plate, vHLOs cultured on the pillar plate exhibited superior maturity, likely due to enhanced nutrient and signaling molecule diffusion. The integration of physiologically relevant patterned liver organoids with the unique pillar plate platform enhanced the capabilities for high-throughput screening and disease modeling.
肝脏组织由肝细胞、胆管细胞、星状细胞、库普弗细胞和窦状内皮细胞组成，源自内胚层和中胚层胚层。通过模拟肝脏的发育过程，已经发表了各种分化方案，以使用诱导多能干细胞（iPSC）生成人肝脏类器官（HLO）。然而，仅源自内胚层胚层的HLO常常遇到技术障碍，如成熟度和功能不足，限制了它们在疾病建模和肝毒性检测中的应用。为了克服这一问题，我们从同一人iPSC系中分别分化出EpCAM内胚层祖细胞（EPC）和中胚层来源的血管祖细胞（VPC）。然后将这些细胞混合在BME-2基质中，并同时分化为血管化人肝脏类器官（vHLO）。值得注意的是，vHLO的成熟度明显高于无血管的HLO，凝血因子分泌增加、白蛋白分泌增加、药物代谢酶（DME）表达增加和胆汁酸转运增加证明了这一点。为了提高检测通量并使vHLO培养小型化，我们在源自EPC和VPC的柱板平台上的BME-2基质中3D生物打印可扩展HLO（eHLO），并与仅源自内胚层的HLO进行比较。与在24孔板中50μL BME-2基质穹顶中培养的HLO相比，在柱板上培养的vHLO表现出更高的成熟度，这可能是由于营养物质和信号分子扩散增强。生理相关的图案化肝脏类器官与独特的柱板平台的整合增强了高通量筛选和疾病建模的能力。
