Mobarra Naser, Soleimani Masoud, Kouhkan Fatemeh, Hesari Zahra, Lahmy Reyhaneh, Mossahebi-Mohammadi Majid, Arefian Ehsan, Jaafarpour Zahra, Nasiri Hajar, Pakzad Reza, Tavakoli Rezvan, Pasalar Parvin
Department of Clinical Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran ; Students'Sciences Research Center, Tehran University of Medical Sciences, Tehran, Iran.
Department of Hematology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
Int J Hematol Oncol Stem Cell Res. 2014 Oct 1;8(4):20-9.
The use of stem cells is considered as an appropriate source in cell therapy and tissue engineering. Differentiation of human induced Pluripotent Stem Cells (hiPSCs) to Hepatocyte-like Cells (HLCs) on mouse embryonic fibroblasts (MEFs) feeders is confronted with several problems that hinder the clinical applications of these differentiated cells for the treatment of liver injuries. Safe appropriate cells for stem cell-based therapies could create new hopes for liver diseases. This work focused on the determination of a capacity/efficiency for the differentiation of the hiPSCs into Hepatocyte-like Cells on a novel human adult bone marrow mesenchymal stem cells (hMSCs) feeder.
Undifferentiated human iPSCs were cultured on mitotically inactivated human adult bone marrow mesenchymal stem cells. A three-step differentiation process has been performed in presence of activin A which added for 3 days to induce a definitive endoderm formation. In the second step, medium was exchanged for six days. Subsequently, cells were treated with oncostatin M plus dexamethasone for 9 days to generate hepatic cells. Endodermic and liver-specific genes were assessed via quantitative reverse transcription-polymerase chain reaction and RT-PCR, moreover, immunocytochemical staining for liver proteins including albumin and alpha-fetoprotein. In addition, functional tests for glycogen storage, oil red examination, urea production and alpha-fetoprotein synthesis, as well as, cells differentiated with a hepatocyte-like morphology was also performed.
Our results show that inactivated human adult bone marrow mesenchymal stem cell feeders could support the efficient differentiation of hiPSCs into HLCs. This process induced differentiation of iPSCs into definitive endocrine cells that expressed sox17, foxa2 and expression of the specific genes profiles in hepatic-like cells. In addition, immunocytochemical analysis confirmed albumin and alpha-fetoprotein protein expression, as well as, the hiPSCs-derived Hepatocyte-like Cells on human feeder exhibited a typical morphology.
we suggested a successful and efficient culture for differentiation and maturation of hepatocytes on an alternative human feeders; this is an important step to generate safe and functional hepatocytes that is vital for regenerative medicine and transplantation on the cell-based therapies.
干细胞的应用被认为是细胞治疗和组织工程的合适细胞来源。在小鼠胚胎成纤维细胞（MEFs）饲养层上，将人诱导多能干细胞（hiPSCs）分化为肝样细胞（HLCs）面临着一些问题，这些问题阻碍了这些分化细胞在肝损伤治疗中的临床应用。基于干细胞的治疗中安全合适的细胞可能为肝脏疾病带来新希望。这项工作聚焦于在新型人成人骨髓间充质干细胞（hMSCs）饲养层上确定hiPSCs向肝样细胞分化的能力/效率。
将未分化的人诱导多能干细胞培养在有丝分裂失活的人成人骨髓间充质干细胞上。在激活素A存在的情况下进行三步分化过程，激活素A添加3天以诱导确定内胚层形成。第二步，更换培养基6天。随后，用抑瘤素M加地塞米松处理细胞9天以生成肝细胞。通过定量逆转录-聚合酶链反应和RT-PCR评估内胚层和肝脏特异性基因，此外，对包括白蛋白和甲胎蛋白在内的肝脏蛋白进行免疫细胞化学染色。另外，还进行了糖原储存、油红检测、尿素生成和甲胎蛋白合成的功能测试，以及对具有肝样形态的分化细胞进行检测。
我们的结果表明，失活的人成人骨髓间充质干细胞饲养层能够支持hiPSCs高效分化为HLCs。这个过程诱导iPSCs分化为表达sox17、foxa2的确定内分泌细胞以及肝样细胞中特定基因谱的表达。此外，免疫细胞化学分析证实了白蛋白和甲胎蛋白的蛋白表达，并且在人饲养层上hiPSCs来源的肝样细胞呈现出典型形态。
我们提出了一种在替代人饲养层上成功且高效地培养肝细胞分化和成熟的方法；这是生成安全且有功能的肝细胞的重要一步，对于再生医学和基于细胞治疗的移植至关重要。
