Health Science and Technologies Interdepartmental Center for Industrial Research (HST-ICIR), University of Bologna;
J Vis Exp. 2021 Mar 10(169). doi: 10.3791/61988.
The main hurdle in developing drug screening techniques for assessing the efficacy of therapeutic strategies in complex diseases is striking a balance between in vitro simplification and recreating the complex in vivo environment, along with the main aim, shared by all screening strategies, of obtaining robust and reliable data, highly predictive for in vivo translation. In the field of demyelinating diseases, the majority of drug screening strategies are based on immortalized cell lines or pure cultures of isolated primary oligodendrocyte precursor cells (OPCs) from newborn animals, leading to strong biases due to the lack of age-related differences and of any real pathological condition or complexity. Here we show the setup of an in vitro system aimed at modeling the physiological differentiation/maturation of neural stem cell (NSC)-derived OPCs, easily manipulated to mimic pathological conditions typical of demyelinating diseases. Moreover, the method includes isolation from fetal and adult brains, giving a system which dynamically differentiates from OPCs to mature oligodendrocytes (OLs) in a spontaneous co-culture which also includes astrocytes. This model physiologically resembles the thyroid hormone-mediated myelination and myelin repair process, allowing the addition of pathological interferents which model disease mechanisms. We show how to mimic the two main components of demyelinating diseases (i.e., hypoxia/ischemia and inflammation), recreating their effect on developmental myelination and adult myelin repair and taking all the cell components of the system into account throughout, while focusing on differentiating OPCs. This spontaneous mixed model, coupled with cell-based high-content screening technologies, allows the development of a robust and reliable drug screening system for therapeutic strategies aimed at combating the pathological processes involved in demyelination and at inducing remyelination.
开发药物筛选技术以评估复杂疾病治疗策略的疗效的主要障碍是在体外简化和再现复杂的体内环境之间取得平衡，同时所有筛选策略的主要目标是获得稳健可靠的数据，对体内转化具有高度预测性。在脱髓鞘疾病领域，大多数药物筛选策略基于永生化细胞系或从新生动物中分离的纯培养物的原代少突胶质前体细胞（OPC），由于缺乏与年龄相关的差异和任何真正的病理条件或复杂性，导致严重的偏差。在这里，我们展示了一种旨在模拟神经干细胞（NSC）衍生的 OPC 生理分化/成熟的体外系统的设置，该系统易于操作，可以模拟脱髓鞘疾病的典型病理条件。此外，该方法包括从胎儿和成人大脑中分离出来，提供了一个系统，该系统可以在自发共培养中从 OPC 动态分化为成熟的少突胶质细胞（OL），其中还包括星形胶质细胞。该模型在生理上类似于甲状腺激素介导的髓鞘形成和髓鞘修复过程，允许添加模拟疾病机制的病理干扰物。我们展示了如何模拟脱髓鞘疾病的两个主要成分（即缺氧/缺血和炎症），再现它们对发育性髓鞘形成和成人髓鞘修复的影响，并在整个过程中考虑系统的所有细胞成分，同时专注于分化的 OPC。这种自发的混合模型，结合基于细胞的高内涵筛选技术，为针对脱髓鞘相关病理过程并诱导髓鞘再生的治疗策略开发了一种稳健可靠的药物筛选系统。
