Conventional immunomarkers stain a fraction of astrocytes in vitro: A comparison of rat cortical and spinal cord astrocytes in naïve and stimulated cultures

In the present study, we analyzed the percent immunopositive cells in astrocyte-enriched, microglia-enriched, and oligodendrocyte-enriched cultures isolated from the cortex or spinal cord of neonatal rats for six markers that have been previously used to label astrocytes: GFAP, GLAST, GLT-1, GS, ALDH1L1, and SOX9. We also stimulated astrocyte cultures with either transforming growth factor (TGF)-β1, shown to induce reactive astrogliosis in rodent cortical astrocytes in vitro, or TGF-β3, shown to dampen reactivity markers in astrocytes in vitro. Markers were evaluated based on the following criteria: (1) ability to stain all astrocytes in a given culture, (2) resistance to changes in the percentage of positively stained astrocytes after stimulation with either TGF-β1 or TGF-β3, and (3) no positive staining of microglia and oligodendrocytes in cultures isolated from the same region in the CNS (cortex or spinal cord). In doing so, we aimed to investigate protein expression differences between cortical and spinal cord astrocytes and changes in marker expression with TGF-β1 and TGF-β3 stimulation in vitro. We found that only SOX9 in cortical cultures and ALDH1L1 in spinal cord cultures labeled more than 75% of the cells in naïve and stimulated astrocyte cultures and stained less than 5% of the cells in microglia and oligodendrocyte cultures. Further, significantly more cortical than spinal cord astrocytes stained for GFAP, GLAST, ALDH1L1, and SOX9 in naïve cultures, whereas significantly more spinal cord than cortical astrocytes stained for GLAST, GS, and ALDH1L1 in TGF-β1-treated cultures. These findings are important as variability in marker staining may lead to misinterpretation of the astrocyte response in cocultures, migration assays, or engineered disease models. The first sheet in the excel file shows the number of CD68+ microglia and MBP+ oligodendrocytes in each culture condition (naïve or stimulated with TGF-β1 or TGF-β3 in cultures derived from either rat cortex or spinal cord), which was used to calculate the percentage of astrocytes in the cultures. The second sheet in the file shows the number of immunopositive cells counted for each replicate in all culture conditions.本研究中，我们针对6种此前用于标记星形胶质细胞（astrocyte）的标志物：GFAP、GLAST、GLT-1、GS、ALDH1L1及SOX9，分析了从新生大鼠皮层或脊髓分离的富集星形胶质细胞、富集小胶质细胞（microglia）及富集少突胶质细胞（oligodendrocyte）培养物中的免疫阳性细胞占比。我们还分别用转化生长因子（TGF）-β1和TGF-β3处理星形胶质细胞培养物：前者已被证实可在体外诱导啮齿类动物皮层星形胶质细胞发生反应性星形胶质化，后者则可在体外抑制星形胶质细胞的反应性标志物表达。本研究基于以下三项标准对标志物进行评估：（1）可特异性染色目标培养物中的全部星形胶质细胞；（2）经TGF-β1或TGF-β3刺激后，免疫阳性星形胶质细胞的占比无显著变化；（3）不会对从中枢神经系统（Central Nervous System, CNS）同一区域分离的小胶质细胞及少突胶质细胞产生阳性染色。本研究旨在借此探究皮层与脊髓星形胶质细胞间的蛋白质表达差异，以及经TGF-β1、TGF-β3体外刺激后星形胶质细胞标志物表达的变化情况。研究结果显示，仅皮层培养物中的SOX9与脊髓培养物中的ALDH1L1，可在未处理及经刺激的星形胶质细胞培养物中标记超过75%的细胞，且在小胶质细胞与少突胶质细胞培养物中的阳性染色细胞占比不足5%。此外，在未处理的培养物中，皮层星形胶质细胞的GFAP、GLAST、ALDH1L1及SOX9阳性染色率显著高于脊髓星形胶质细胞；而在经TGF-β1处理的培养物中，脊髓星形胶质细胞的GLAST、GS及ALDH1L1阳性染色率则显著高于皮层星形胶质细胞。本研究结果具有重要意义，因为标志物染色的差异可能会导致共培养、迁移实验或工程化疾病模型中星形胶质细胞反应的解读出现偏差。本Excel文件的第一张工作表展示了各培养条件下（即源自新生大鼠皮层或脊髓的未处理培养物，或经TGF-β1、TGF-β3刺激的培养物）CD68+小胶质细胞与MBP+少突胶质细胞的数量，该数据用于计算培养物中星形胶质细胞的占比。该文件的第二张工作表则展示了所有培养条件下，各生物学重复的免疫阳性细胞计数结果。