Department of Biomedical Sciences of Cells and Systems, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.
Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Naples, Italy.
J Hepatol. 2019 Aug;71(2):344-356. doi: 10.1016/j.jhep.2019.03.031. Epub 2019 Apr 6.
BACKGROUND & AIMS: Hepatocyte polarity is essential for the development of bile canaliculi and for safely transporting bile and waste products from the liver. Functional studies of autologous mutated proteins in the context of the polarized hepatocyte have been challenging because of the lack of appropriate cell models. The aims of this study were to obtain a patient-specific hepatocyte model that recapitulated hepatocyte polarity and to employ this model to study endogenous mutant proteins in liver diseases that involve hepatocyte polarity.
Urine cell-derived pluripotent stem cells, taken from a patient with a homozygous mutation in ATP7B and a patient with a heterozygous mutation, were differentiated towards hepatocyte-like cells (hiHeps). HiHeps were also derived from a patient with MEDNIK syndrome.
Polarized hiHeps that formed in vivo-like bile canaliculi could be generated from embryonic and patient urine cell-derived pluripotent stem cells. HiHeps recapitulated polarized protein trafficking processes, exemplified by the Cu-induced redistribution of the copper transporter protein ATP7B to the bile canalicular domain. We demonstrated that, in contrast to the current dogma, the most frequent yet enigmatic Wilson disease-causing ATP7B-H1069Q mutation per se did not preclude trafficking of ATP7B to the trans-Golgi Network. Instead, it prevented its Cu-induced polarized redistribution to the bile canalicular domain, which could not be reversed by pharmacological folding chaperones. Finally, we demonstrate that hiHeps from a patient with MEDNIK syndrome, suffering from liver copper overload of unclear etiology, showed no defect in the Cu-induced redistribution of ATP7B to the bile canaliculi.
Functional cell polarity can be achieved in patient pluripotent stem cell-derived hiHeps, enabling, for the first time, the study of the endogenous mutant proteins, patient-specific pathogenesis and drug responses for diseases where hepatocyte polarity is a key factor.
This study demonstrates that cells that are isolated from urine can be reprogrammed in a dish towards hepatocytes that display architectural characteristics similar to those seen in the intact liver. The application of this methodology to cells from patients diagnosed with inherited copper metabolism-related liver diseases (that is, Wilson disease and MEDNIK syndrome) revealed unexpected and novel insights into patient mutation-specific disease mechanisms and drug responses.
肝细胞极性对于胆汁 canaliculi 的发育以及安全地将胆汁和废物从肝脏中运输至关重要。由于缺乏适当的细胞模型，在极化的肝细胞背景下对自体突变蛋白进行功能研究一直具有挑战性。本研究的目的是获得能够重现肝细胞极性的患者特异性肝细胞模型，并利用该模型研究涉及肝细胞极性的肝脏疾病中的内源性突变蛋白。
从一名 ATP7B 纯合突变和一名杂合突变患者的尿液细胞衍生的多能干细胞中获取细胞，将其分化为肝细胞样细胞（hiHeps）。hiHeps 也来自 MEDNIK 综合征患者。
来自胚胎和患者尿液细胞衍生的多能干细胞的可形成类似体内胆汁 canaliculi 的极化 hiHeps 可以被生成。hiHeps 重现了极化蛋白运输过程，例如铜诱导的铜转运蛋白 ATP7B 向胆汁 canaliculi 域的重新分布。我们证明，与当前的教条相反，最常见但仍令人费解的威尔逊病致病 ATP7B-H1069Q 突变本身并没有阻止 ATP7B 向反式高尔基体网络的运输。相反，它阻止了铜诱导的向胆汁 canaliculi 域的极化再分布，这一过程不能被药理学折叠伴侣逆转。最后，我们证明，来自 MEDNIK 综合征患者的 hiHeps 中，ATP7B 向胆汁 canaliculi 的铜诱导再分布没有缺陷，而 MEDNIK 综合征患者的肝脏铜过载病因不明。
患者多能干细胞衍生的 hiHeps 中可以实现功能性细胞极性，首次能够研究内源性突变蛋白、患者特异性发病机制和药物反应，这些对于肝细胞极性是关键因素的疾病非常重要。
本研究表明，从尿液中分离的细胞可以在培养皿中被重新编程为具有与完整肝脏相似的结构特征的肝细胞。将这种方法应用于诊断为遗传性铜代谢相关肝脏疾病（即威尔逊病和 MEDNIK 综合征）的患者的细胞中，揭示了对患者突变特异性疾病机制和药物反应的意外和新的见解。
