Effective three-dimensional culture of hepatocyte-like cells generated from human adipose-derived mesenchymal stem cells
Effective three-dimensional culture of hepatocyte-like cells generated from human adipose-derived mesenchymal stem cells
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DOI：
10.1002/jhbp.1024
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发表时间：
2021-08-10
影响因子：
3
通讯作者：
Shimada, Mitsuo
中科院分区：
文献类型：
--
作者：
Saito, Yu;Ikemoto, Tetsuya;Shimada, Mitsuo
背景本研究的目的是阐明一种新的三维（3D）培养系统对从人脂肪间充质干细胞（ADSC）产生的肝细胞样细胞（HLC）的有效性。方法将每孔含或不含0.1mg/mL人重组肽μ片段的人ADSCs（2 × 10（4））接种于96孔U形底板中，然后应用我们的三步分化方案21天。在每个步骤中，研究细胞形态和基因表达。在HLC分化后评价成熟肝细胞功能。这些参数之间进行了比较2D和3D培养的HLCs，并进行了DNA微阵列分析。最后，将HLCs移植到CCl 4诱导的急性肝衰竭模型小鼠体内。结果二维培养21 d的HLCs不再呈梭形，第6d开始形成球状体，三维培养的HLCs球状体逐渐增大。在3D培养组中，连续性内胚层、成肝细胞和肝细胞基因表现出显著更高的表达。三维培养的HLCs也有更高的白蛋白表达，CYP 3A 4活性，尿素合成和铵代谢，以及更高的离子转运蛋白，凝血和细胞通讯基因的表达。HLC移植可改善血清肝功能，尤其是T-Bil水平，并移植到HLA I类阳性的免疫缺陷小鼠中。结论新的三维培养方法能有效提高肝细胞功能。我们的HLCs可能有希望用于临床细胞移植治疗代谢性疾病。
Background The aim of this study was to clarify the effectiveness of a new three-dimensional (3D) culture system for hepatocyte-like cells (HLCs) generated from human adipose-derived mesenchymal stem cells (ADSCs). Methods Human ADSCs (2 x 10(4)) with or without 0.1 mg/mL human recombinant peptide mu-piece per well were seeded in a 96-well U-bottom plate and then our three-step differentiation protocol was applied for 21 days. At each step, cell morphology and gene expression were investigated. Mature hepatocyte functions were evaluated after HLC differentiation. These parameters were compared between 2D- and 3D-cultured HLCs, and, DNA microarray analysis was also performed. Finally, HLCs were transplanted in to CCl4 induced acute liver failure model mice. Results Two-dimensional-cultured HLCs at day 21 did not have a spindle shape and had formed spheroids after day 6, which gradually increased in size for 3D-cultured HLCs. Definitive endoderm, hepatoblast, and hepatocyte genes showed significantly higher expression in the 3D culture group. Three-dimensional-cultured HLCs also had higher albumin expression, CYP3A4 activity, urea synthesis, and ammonium metabolism, and much higher expression of ion transporter, blood coagulation, and cell communication genes. HLC transplantation improved serum liver function, especially in T-Bil levels, and engrafted into immunodeficient mice with HLA class I positive staining. Conclusion Our new 3D culture protocol is effective to improve hepatocyte functions. Our HLCs might be promising for clinical cell transplantation to treat metabolic disease.