Trindade Pablo, Loiola Erick Correia, Gasparotto Juciano, Ribeiro Camila Tiefensee, Cardozo Pablo Leal, Devalle Sylvie, Salerno José Alexandre, Ornelas Isis Moraes, Ledur Pitia Flores, Ribeiro Fabiola Mara, Ventura Ana Lucia Marques, Moreira José Claudio Fonseca, Gelain Daniel Pens, Porciúncula Lisiane Oliveira, Rehen Stevens Kastrup
Instituto D'Or de Pesquisa e Ensino (IDOR), Rio de Janeiro, Brazil.
Pós-Graduação em Biologia Molecular e Celular, Universidade Federal do Estado do Rio de Janeiro, Rio de Janeiro, Brazil.
Glia. 2020 Jul;68(7):1396-1409. doi: 10.1002/glia.23786. Epub 2020 Jan 31.
Astrogliosis comprises a variety of changes in astrocytes that occur in a context-specific manner, triggered by temporally diverse signaling events that vary with the nature and severity of brain insults. However, most mechanisms underlying astrogliosis were described using animals, which fail to reproduce some aspects of human astroglial signaling. Here, we report an in vitro model to study astrogliosis using human-induced pluripotent stem cells (iPSC)-derived astrocytes which replicate temporally intertwined aspects of reactive astrocytes in vivo. We analyzed the time course of astrogliosis by measuring nuclear translocation of NF-kB, production of cytokines, changes in morphology and function of iPSC-derived astrocytes exposed to TNF-α. We observed NF-kB p65 subunit nuclear translocation and increased gene expression of IL-1β, IL-6, and TNF-α in the first hours following TNF-α stimulation. After 24 hr, conditioned media from iPSC-derived astrocytes exposed to TNF-α exhibited increased secretion of inflammation-related cytokines. After 5 days, TNF-α-stimulated cells presented a typical phenotype of astrogliosis such as increased immunolabeling of Vimentin and GFAP and nuclei with elongated shape and shrinkage. Moreover, ~50% decrease in aspartate uptake was observed during the time course of astrogliosis with no evident cell damage, suggesting astroglial dysfunction. Together, our results indicate that human iPSC-derived astrocytes reproduce canonical events associated with astrogliosis in a time dependent fashion. The approach described here may contribute to a better understanding of mechanisms governing human astrogliosis with potential applicability as a platform to uncover novel biomarkers and drug targets to prevent or mitigate astrogliosis associated with human brain disorders.
星形胶质细胞增生包括星形胶质细胞的多种变化，这些变化以特定背景的方式发生，由随脑损伤的性质和严重程度而变化的不同时间的信号事件触发。然而，大多数关于星形胶质细胞增生的机制是在动物身上描述的，而动物无法重现人类星形胶质细胞信号传导的某些方面。在这里，我们报告了一种体外模型，使用人诱导多能干细胞（iPSC）衍生的星形胶质细胞来研究星形胶质细胞增生，这些细胞在体内复制了反应性星形胶质细胞在时间上相互交织的方面。我们通过测量NF-κB的核转位、细胞因子的产生、暴露于TNF-α的iPSC衍生星形胶质细胞的形态和功能变化来分析星形胶质细胞增生的时间进程。我们观察到在TNF-α刺激后的最初几个小时内，NF-κB p65亚基发生核转位，IL-1β、IL-6和TNF-α的基因表达增加。24小时后，暴露于TNF-α的iPSC衍生星形胶质细胞的条件培养基显示炎症相关细胞因子的分泌增加。5天后，TNF-α刺激的细胞呈现出星形胶质细胞增生的典型表型，如波形蛋白和胶质纤维酸性蛋白（GFAP）的免疫标记增加，细胞核呈细长形且缩小。此外，在星形胶质细胞增生的时间进程中观察到天冬氨酸摄取减少约50%，且无明显细胞损伤，提示星形胶质细胞功能障碍。总之，我们的结果表明，人iPSC衍生的星形胶质细胞以时间依赖性方式重现了与星形胶质细胞增生相关的典型事件。这里描述的方法可能有助于更好地理解控制人类星形胶质细胞增生的机制，并有可能作为一个平台来发现新的生物标志物和药物靶点，以预防或减轻与人类脑部疾病相关的星形胶质细胞增生。
