Accelerated differentiation of human induced pluripotent stem cells to blood-brain barrier endothelial cells.
Accelerated differentiation of human induced pluripotent stem cells to blood-brain barrier endothelial cells.
复制标题
DOI：
10.1186/s12987-017-0059-0
复制
发表时间：
2017-04-13
影响因子：
7.3
通讯作者：
Lippmann ES
中科院分区：
文献类型：
--
作者：
Hollmann EK;Bailey AK;Potharazu AV;Neely MD;Bowman AB;Lippmann ES
由于它们能够无限增殖和专门化成几乎任何细胞类型，人类诱导多能干细胞（iPSC）为产生人脑微血管内皮细胞（BMEC）提供了前所未有的机会，这些细胞构成了血脑屏障（BBB），用于研究目的。不幸的是，将iPSC分化为纯化的BMEC所需的时间、费用和专业知识阻碍了它们的广泛应用。在这里，我们报告了使用一种确定的培养基，该培养基加速iPSC向BMEC的分化，同时实现与通过已建立的方法产生的BMEC相当的性能。将诱导的多能干细胞以确定的密度接种，并使用称为E6的确定培养基分化为BMEC。通过跨内皮电阻（TEER）、荧光素渗透性、P-糖蛋白和MRP家族外排转运蛋白活性评估所得纯化BMEC表型。内皮标志物和它们的签名紧密连接蛋白的表达用免疫细胞化学证实。通过TEER测量评估与星形胶质细胞和周细胞共培养对纯化的BMEC的影响。在独立的iPSC系中证实了分化方法的稳健性。使用E6培养基，加上更新的培养方法，将iPSC分化为BMEC的时间从13天缩短到8天。E6来源的BMEC表达GLUT-1、claudin-5、occludin、PECAM-1和VE-钙粘蛋白，并且在多个iPSC系中始终达到超过2500 Ω × cm 2的TEER值，最大TEER值为4678 ± 49 Ω × cm 2，荧光素渗透率低于1.95 × 10−7 cm/s。E6衍生的BMEC在至少8天内保持TEER高于1000 Ω × cm 2，与通过常规方法分化的BMEC相比，外排转运蛋白活性无统计学差异。还发现该方法支持携带与帕金森病相关的双等位基因PARK 2突变的BMEC的长期稳定性。最后，使用E6培养基分化的BMEC对来自星形胶质细胞和周细胞的诱导线索作出响应，并达到6635 ± 315 Ω × cm 2的最大TEER值，据我们所知，这是迄今为止报道的最高体外TEER值。考虑到加速分化，等效性能和降低生产BMEC的成本，我们更新的方法应该使iPSC衍生的体外BBB模型更容易用于各种应用。本文的在线版本（doi：10.1186/s12987-017-0059-0）包含补充材料，可供授权用户使用。
Due to their ability to limitlessly proliferate and specialize into almost any cell type, human induced pluripotent stem cells (iPSCs) offer an unprecedented opportunity to generate human brain microvascular endothelial cells (BMECs), which compose the blood–brain barrier (BBB), for research purposes. Unfortunately, the time, expense, and expertise required to differentiate iPSCs to purified BMECs precludes their widespread use. Here, we report the use of a defined medium that accelerates the differentiation of iPSCs to BMECs while achieving comparable performance to BMECs produced by established methods. Induced pluripotent stem cells were seeded at defined densities and differentiated to BMECs using defined medium termed E6. Resultant purified BMEC phenotypes were assessed through trans-endothelial electrical resistance (TEER), fluorescein permeability, and P-glycoprotein and MRP family efflux transporter activity. Expression of endothelial markers and their signature tight junction proteins were confirmed using immunocytochemistry. The influence of co-culture with astrocytes and pericytes on purified BMECs was assessed via TEER measurements. The robustness of the differentiation method was confirmed across independent iPSC lines. The use of E6 medium, coupled with updated culture methods, reduced the differentiation time of iPSCs to BMECs from thirteen to 8 days. E6-derived BMECs expressed GLUT-1, claudin-5, occludin, PECAM-1, and VE-cadherin and consistently achieved TEER values exceeding 2500 Ω × cm2 across multiple iPSC lines, with a maximum TEER value of 4678 ± 49 Ω × cm2 and fluorescein permeability below 1.95 × 10−7 cm/s. E6-derived BMECs maintained TEER above 1000 Ω × cm2 for a minimum of 8 days and showed no statistical difference in efflux transporter activity compared to BMECs differentiated by conventional means. The method was also found to support long-term stability of BMECs harboring biallelic PARK2 mutations associated with Parkinson’s Disease. Finally, BMECs differentiated using E6 medium responded to inductive cues from astrocytes and pericytes and achieved a maximum TEER value of 6635 ± 315 Ω × cm2, which to our knowledge is the highest reported in vitro TEER value to date. Given the accelerated differentiation, equivalent performance, and reduced cost to produce BMECs, our updated methods should make iPSC-derived in vitro BBB models more accessible for a wide variety of applications. The online version of this article (doi:10.1186/s12987-017-0059-0) contains supplementary material, which is available to authorized users.