Aravalli Rajagopal N, Collins Daniel P, Hapke Joel H, Crane Andrew T, Steer Clifford J
Department of Electrical and Computer Engineering, University of Minnesota, Minneapolis, MN 55455, USA.
Cytomedical Design Group LLC, St. Paul, MN 55127, USA.
Hepat Med. 2020 Feb 13;12:15-27. doi: 10.2147/HMER.S243277. eCollection 2020.
Primary human hepatocytes (PHHs) are the ideal candidates for studying critical liver functions such as drug metabolism and toxicity. However, as they are isolated from discarded livers that are unsuitable for transplantation, they possess limited expansion ability and their enzymatic functions deteriorate rapidly because they are often of poor quality. Therefore, there is a compelling reason to find reliable alternative sources of hepatocytes.
In this study, we report on efficient and robust differentiation of embryonic stem cells (ESC) from the common marmoset into functional hepatocyte-like cells (HLC) using a simple, and reproducible three-step procedure. ESC-derived HLCs were examined by morphological analysis and tested for their expression of hepatocyte-specific markers using a combination of immunohistochemistry, RT-PCR, and biochemical assays. Primary human hepatocytes were used as controls.
ESC-derived HLCs expressed each of the hepatocyte-specific markers tested, including albumin; α-fetoprotein; asialoglycoprotein receptor 1; α-1 antitrypsin; hepatocyte nuclear factors 1α and 4; cytokeratin 18; hepatocyte growth factor receptor; transferrin; tyrosine aminotransferase; alkaline phosphatase; c-reactive protein; cytochrome P450 enzymes CYP1A2, CYP2E1 and CYP3A4; and coagulation factors FVII and FIX. They were functionally competent as demonstrated by biochemical assays in addition to producing urea.
Our data strongly suggest that marmoset HLCs possess characteristics similar to those of PHHs. They could, therefore, be invaluable for studies on drug metabolism and cell transplantation therapy for a variety of liver disorders. Because of the similarities in the anatomical and physiological features of the common marmoset to that of humans, is an appropriate animal model to study human disease conditions and cellular functions.
原代人肝细胞（PHH）是研究药物代谢和毒性等关键肝功能的理想细胞。然而，由于它们是从不适用于移植的废弃肝脏中分离出来的，其增殖能力有限，且酶功能因质量往往较差而迅速衰退。因此，迫切需要找到可靠的肝细胞替代来源。
在本研究中，我们报告了使用一种简单且可重复的三步程序，将普通狨猴的胚胎干细胞（ESC）高效且稳定地分化为功能性肝样细胞（HLC）。通过形态学分析对ESC来源的HLC进行检查，并结合免疫组织化学、逆转录-聚合酶链反应（RT-PCR）和生化分析检测其肝细胞特异性标志物的表达。以原代人肝细胞作为对照。
ESC来源的HLC表达了所检测的每种肝细胞特异性标志物，包括白蛋白、甲胎蛋白、去唾液酸糖蛋白受体1、α-1抗胰蛋白酶、肝细胞核因子1α和4、细胞角蛋白18、肝细胞生长因子受体、转铁蛋白、酪氨酸转氨酶、碱性磷酸酶、C反应蛋白、细胞色素P450酶CYP1A2、CYP2E1和CYP3A4以及凝血因子FVII和FIX。除了产生尿素外，生化分析表明它们具有功能活性。
我们的数据有力地表明，狨猴HLC具有与PHH相似的特征。因此，它们对于药物代谢研究和多种肝脏疾病的细胞移植治疗可能具有极高价值。由于普通狨猴在解剖学和生理学特征上与人类相似，是研究人类疾病状况和细胞功能的合适动物模型。
