Optimized protocol for the hepatic differentiation of induced pluripotent stem cells in a fluidic microenvironment
Optimized protocol for the hepatic differentiation of induced pluripotent stem cells in a fluidic microenvironment
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DOI：
10.1002/bit.26970
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发表时间：
2019-07-01
影响因子：
3.8
通讯作者：
Leclerc, Eric
中科院分区：
文献类型：
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作者：
Danoy, Mathieu;Bernier, Myriam Lereau;Leclerc, Eric
在本研究中，我们评估了微流控生物芯片中人诱导多能干细胞（hiPSC）肝分化的不同方案的性能。测试了完全和部分片上差异化的策略。与完全芯片上分化不同，在分化过程中，iPSC 从培养皿转移到生物芯片会产生异质组织，与培养皿中的对照培养物相比，其肝脏特征增强。生物芯片中的组织由表达稳定蛋白 1 或白蛋白的细胞组成，而在对照中未检测到稳定蛋白 1。功能分析还显示生物芯片中白蛋白的生产率翻了一番（每 10(6) 细胞每天约 2,000 ng）。除此之外，生物芯片和对照中获得的组织表现出特定胆汁酸的代谢。使用nanoCAGE进行的全转录组分析显示对照培养物和生物芯片培养物之间存在302个基因的差异表达，生物芯片中的肝脏分化程度较高，并且典型肝脏转录因子的启动子基序活性增加，例如雌激素相关受体α（ESRRA）、肝核因子1（HNF1A）、肝核因子4（HNF4A）、转录因子4（TCF4）和CCAAT增强子结合蛋白α （CEBPA）。基因集富集分析确定了与细胞外基质、组织重组、缺氧诱导转录因子和糖酵解相关的几种途径，这些途径在生物芯片培养物中受到差异调节。然而，CK19/ALB 阳性细胞的存在和培养物中测量的甲胎蛋白水平仍然反映了原始的分化模式。总体而言，我们确定了改善芯片上肝脏分化的关键参数，包括肝祖细胞的成熟阶段、接种密度、粘附时间和灌注流速。这些参数的优化进一步导致建立了一种在微流体生物芯片中将 hiPSC 可重复分化为肝细胞样细胞的方案，与培养皿培养相比具有显着改进。
In the present study, we evaluated the performance of different protocols for the hepatic differentiation of human-induced pluripotent stem cells (hiPSCs) in microfluidic biochips. Strategies for complete and partial on-chip differentiation were tested. Unlike full on-chip differentiation, the transfer of iPSCs from Petri dishes to biochips during the differentiation process produced a heterogeneous tissue with enhanced hepatic features compared with control cultures in Petri dishes. The tissue in biochips was constituted of cells expressing either stabilin-1 or albumin, while no stabilin-1 was detected in controls. Functional analysis also revealed double the production rate for albumin in biochips (about 2,000 ng per day per 10(6) cells). Besides this, tissues obtained in biochips and controls exhibited the metabolism of a specific bile acid. Whole transcriptome analysis with nanoCAGE exhibited a differential expression of 302 genes between control and biochip cultures and a higher degree of hepatic differentiation in biochips, together with increased promoter motif activity for typical liver transcription factors such as estrogen related receptor alpha (ESRRA), hepatic nuclear factor 1 (HNF1A), hepatic nuclear factor 4 (HNF4A), transcription factor 4 (TCF4), and CCAAT enhancer binding protein alpha (CEBPA). Gene set enrichment analysis identified several pathways related to the extracellular matrix, tissue reorganization, hypoxia-inducible transcription factor, and glycolysis that were differentially modulated in biochip cultures. However, the presence of CK19/ALB-positive cells and the -fetoprotein levels measured in the cultures still reflect primitive differentiation patterns. Overall, we identified key parameters for improved hepatic differentiation on-chip, including the maturation stage of hepatic progenitors, inoculation density, adhesion time, and perfusion flow rate. Optimization of these parameters further led to establish a protocol for reproducible differentiation of hiPSCs into hepatocyte-like cells in microfluidic biochips with significant improvements over Petri dish cultures.