Studying the Inflammatory Responses to Amyloid Beta Oligomers in Brain-Specific Pericyte and Endothelial Co-Culture From Human Stem Cells
Studying the Inflammatory Responses to Amyloid Beta Oligomers in Brain-Specific Pericyte and Endothelial Co-Culture From Human Stem Cells
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DOI：
10.3389/fceng.2022.927188
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发表时间：
2022-07
期刊：
影响因子：
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通讯作者：
Mark Marzano;Xingchi Chen;Teal A. Russell;Angelica Medina;Zizheng Wang;Timothy Hua;Changchun Zeng;Xueju Wang;Q. Sang;Hengli Tang;Y. Yun;Yan Li
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文献类型：
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作者：
Mark Marzano;Xingchi Chen;Teal A. Russell;Angelica Medina;Zizheng Wang;Timothy Hua;Changchun Zeng;Xueju Wang;Q. Sang;Hengli Tang;Y. Yun;Yan Li
背景：近年来，基于人多能干细胞的体外血脑屏障（BBB）模型因其与中枢神经系统健康的关系而在治疗学中受到广泛关注。为了更好地了解血脑屏障的特性，以及它如何对炎症刺激做出反应，如何通过靶向或非靶向细胞治疗，更具体地说是细胞外囊泡，建立准确的体外血脑屏障模型是至关重要的。方法：使用双SMAD信号抑制剂和Wnt激活加成纤维细胞生长因子2 (FGF-2)，从iPSK3细胞分化成脑特异性周细胞（iPCs）。通过免疫染色、流式细胞术和RT-PCR对衍生细胞进行表征。同时，使用Wnt激活、BMP4、FGF2、VEGF和SB431542衍生血管类器官。复制类器官，并用维甲酸处理，以增强分化的脑内皮细胞的血脑屏障（BBB）特征。在transwell系统和3D微流体通道中对iPCs和iec进行共培养。结果：衍生的iPCs表达常见标记物PDGFRb和NG2，以及脑特异性基因FOXF2、ABCC9、KCNJ8和ZIC1。衍生的iECs表达常见的内皮细胞标志物CD31、VE-cadherin和bbb相关基因BRCP、GLUT-1、PGP、ABCC1、OCLN和SLC2A1。两种细胞类型的共培养通过上调TNFa、IL6、NFKB、Casp3、SOD2和TP53的表达来响应淀粉样蛋白β42低聚物的刺激。共培养还显示出跨内皮电阻的特性。在一个基于三维微流控技术的设备上演示了血管化策略的概念验证。结论：衍生的iPCs和iECs具有脑特异性，iPCs和iECs共培养提供了具有炎症反应的体外血脑屏障模型。本研究对建立神经系统疾病模型和药物筛选的微生理系统具有重要意义。
Background: Recently, the in vitro blood–brain barrier (BBB) models derived from human pluripotent stem cells have been given extensive attention in therapeutics due to the implications they have with the health of the central nervous system. It is essential to create an accurate BBB model in vitro in order to better understand the properties of the BBB, and how it can respond to inflammatory stimulation and be passed by targeted or non-targeted cell therapeutics, more specifically extracellular vesicles. Methods: Brain-specific pericytes (iPCs) were differentiated from iPSK3 cells using dual SMAD signaling inhibitors and Wnt activation plus fibroblast growth factor 2 (FGF-2). The derived cells were characterized by immunostaining, flow cytometry, and RT-PCR. In parallel, blood vessels organoids were derived using Wnt activation, BMP4, FGF2, VEGF, and SB431542. The organoids were replated and treated with retinoic acid to enhance the blood–brain barrier (BBB) features in the differentiated brain endothelial cells (iECs). Co-culture was performed for iPCs and iECs in the transwell system and 3D microfluidics channels. Results: The derived iPCs expressed common markers PDGFRb and NG2, and brain-specific genes FOXF2, ABCC9, KCNJ8, and ZIC1. The derived iECs expressed common endothelial cell markers CD31, VE-cadherin, and BBB-associated genes BRCP, GLUT-1, PGP, ABCC1, OCLN, and SLC2A1. The co-culture of the two cell types responded to the stimulation of amyloid β42 oligomers by the upregulation of the expression of TNFa, IL6, NFKB, Casp3, SOD2, and TP53. The co-culture also showed the property of trans-endothelial electrical resistance. The proof of concept vascularization strategy was demonstrated in a 3D microfluidics-based device. Conclusion: The derived iPCs and iECs have brain-specific properties, and the co-culture of iPCs and iECs provides an in vitro BBB model that show inflammatory response. This study has significance in establishing micro-physiological systems for neurological disease modeling and drug screening.