Induced pluripotent stem cell-derived microglia (iMGL) represent an excellent tool in studying microglial function in health and disease. Yet, since differentiation and survival of iMGL are highly reliant on colony-stimulating factor 1 receptor (CSF1R) signaling, it is difficult to use iMGL to study microglial dysfunction associated with pathogenic defects in CSF1R. Serial modifications to an existing iMGL protocol were made, including but not limited to changes in growth factor combination to drive microglial differentiation, until successful derivation of microglia-like cells from an adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) patient carrying a c.2350G > A (p.V784M) CSF1R variant. Using healthy control lines, the quality of the new iMGL protocol was validated through cell yield assessment, measurement of microglia marker expression, transcriptomic comparison to primary microglia, and evaluation of inflammatory and phagocytic activities. Similarly, molecular and functional characterization of the ALSP patient-derived iMGL was carried out in comparison to healthy control iMGL. The newly devised protocol allowed the generation of iMGL with enhanced transcriptomic similarity to cultured primary human microglia and with higher scavenging and inflammatory competence at ~ threefold greater yield compared to the original protocol. Using this protocol, decreased CSF1R autophosphorylation and cell surface expression was observed in iMGL derived from the ALSP patient compared to those derived from healthy controls. Additionally, ALSP patient-derived iMGL presented a migratory defect accompanying a temporal reduction in purinergic receptor P2Y12 (P2RY12) expression, a heightened capacity to internalize myelin, as well as heightened inflammatory response to Pam3CSK4. Poor P2RY12 expression was confirmed to be a consequence of CSF1R haploinsufficiency, as this feature was also observed following CSF1R knockdown or inhibition in mature control iMGL, and in CSF1RWT/KO and CSF1RWT/E633K iMGL compared to their respective isogenic controls. We optimized a pre-existing iMGL protocol, generating a powerful tool to study microglial involvement in human neurological diseases. Using the optimized protocol, we have generated for the first time iMGL from an ALSP patient carrying a pathogenic CSF1R variant, with preliminary characterization pointing toward functional alterations in migratory, phagocytic and inflammatory activities.

中文翻译：

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从干细胞中衍生小胶质细胞的适应方案及其在 CSF1R 相关疾病研究中的应用


诱导多能干细胞衍生的小胶质细胞（iMGL）是研究小胶质细胞在健康和疾病中的功能的绝佳工具。然而，由于 iMGL 的分化和存活高度依赖于集落刺激因子 1 受体 (CSF1R) 信号传导，因此很难使用 iMGL 来研究与 CSF1R 致病性缺陷相关的小胶质细胞功能障碍。对现有的 iMGL 方案进行了一系列修改，包括但不限于改变生长因子组合以驱动小胶质细胞分化，直到从成人发病的轴突球体和色素胶质细胞 (ALSP) 患者成功衍生出小胶质细胞样细胞c.2350G > A (p.V784M) CSF1R 变体。使用健康对照系，通过细胞产量评估、小胶质细胞标记物表达测量、与原代小胶质细胞的转录组比较以及炎症和吞噬活性评估来验证新 iMGL 方案的质量。同样，对 ALSP 患者来源的 iMGL 进行分子和功能表征，并与健康对照 iMGL 进行比较。新设计的方案允许生成 iMGL，其与培养的原代人类小胶质细胞具有增强的转录组相似性，并且具有更高的清除和炎症能力，与原始方案相比，产量提高了约三倍。使用该方案，与来自健康对照的 iMGL 相比，在来自 ALSP 患者的 iMGL 中观察到 CSF1R 自磷酸化和细胞表面表达减少。 此外，ALSP 患者来源的 iMGL 存在迁移缺陷，伴随嘌呤能受体 P2Y12 (P2RY12) 表达暂时减少、内化髓磷脂的能力增强以及对 Pam3CSK4 的炎症反应增强。 P2RY12 表达差被证实是 CSF1R 单倍体不足的结果，因为在成熟对照 iMGL 中以及 CSF1RWT/KO 和 CSF1RWT/E633K iMGL 中与各自的同基因对照相比，在 CSF1R 敲低或抑制后也观察到了这一特征。我们优化了现有的 iMGL 协议，生成了一个强大的工具来研究小胶质细胞与人类神经系统疾病的关系。使用优化的方案，我们首次从携带致病性 CSF1R 变异的 ALSP 患者中生成 iMGL，初步表征表明迁移、吞噬和炎症活动的功能改变。