246.8: In Vivo Monitoring of Liver Organoids Differentiation Using Live-Screening-Optical Technologies
246.8: In Vivo Monitoring of Liver Organoids Differentiation Using Live-Screening-Optical Technologies
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246.8：使用实时筛选光学技术体内监测肝脏类器官分化
DOI：
10.1097/01.tp.0000886176.20263.10
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发表时间：
2022
期刊：
影响因子：
6.2
通讯作者：
Perelman, Lev T.
中科院分区：
文献类型：
--
作者：
Pettinato, Giuseppe;Coughlan, Mark F.;Zhang, Xuejun;Chen, Liming;Glyavina, Maria;Zakharov, Yuri N.;Zhang, Lei;Qiu, Le;Fisher, Robert A.;Perelman, Lev T.
方法：利用 HLA-II 类敲除 hiPSC 与内皮细胞混合，利用我们的 ROCKi/Spin-free 技术 8 实现人胚状体 (hEB) 的生成。使用共焦光散射光谱显微镜技术，我们监测了 hEB 在整个分化过程中的染色质重塑。该分析使我们能够根据观察到的全基因组修饰来表征我们的四阶段肝细胞分化方案。我们还进行了经典生化分析，以确认通过我们的实时筛选光学技术获得的结果。结果：分化肝脏类器官后，进行实时染色质重塑，以显示分化过程中染色质结构（常染色质与异染色质）的变化（图 1 A 和 B）。通过实时 PCR 检测到几种肝脏标记物以及凝血因子的基因表达，显示我们的肝脏类器官的成熟进展。结论：实时监测我们分化的肝脏类器官是评估活细胞分化进展的一个很好的工具。我们的数据证明了共焦光散射光谱显微镜能够提供肝脏分化过程中体内分化状态的关键信息，为临床转化提供了必要的新工具。
Methods: The generation of human embryoid bodies (hEBs) was achieved utilizing HLA-Class II knockout hiPSCs mixed with endothelial cells, employing our ROCKi/Spin-free technology 8. Using confocal light scattering spectroscopic microscopy technology we monitored the chromatin remodeling of our hEBs during the whole differentiation process. This analysis allowed us to characterize our four-stage hepatocyte differentiation protocol according to the whole genome modifications observed. We also carried out classical biochemical analyses to confirm results obtained with our live-screening-optical technologies.Results: Differentiating liver organoids were followed by performing real-time chromatin remodeling to show changes in chromatin structure (euchromatin vs heterochromatin) over the course of the differentiation process (Fig. 1 A and B). Gene expression of several hepatic markers, as well as coagulation factors, were detected by real-time PCR showing a progression in the maturation of our liver organoids.Conclusions: Real-time monitoring of our differentiating liver organoids represents a great tool to evaluate the differentiation advancement in live cells. Our data demonstrated the ability of confocal light scattering spectroscopic microscopy to provide critical information of the differentiation status in vivo during liver differentiation, providing a new tool necessary for the progression to clinical translation.