Diabetes Research, InSphero AG, Schlieren, Switzerland.
Department of Cardio-Metabolic Diseases, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riß, Germany.
Front Endocrinol (Lausanne). 2022 Jul 4;13:854094. doi: 10.3389/fendo.2022.854094. eCollection 2022.
Restoration of β-cell mass through the induction of proliferation represents an attractive therapeutic approach for the treatment of diabetes. However, intact and dispersed primary islets suffer from rapidly deteriorating viability and function , posing a significant challenge for their experimental use in proliferation studies. Here, we describe a novel method for the assessment of compound effects on β-cell proliferation and count using reaggregated primary human islets, or islet microtissues (MTs), which display homogeneous size and tissue architecture as well as robust and stable functionality and viability for 4 weeks in culture. We utilized this platform to evaluate the dose-dependent short- and long-term effects of harmine on β-cell proliferation and function. Following compound treatment and EdU incorporation, islet MTs were stained and confocal-imaged for DAPI (nuclear marker), NKX6.1 (β-cell marker), and EdU (proliferation marker), allowing automated 3D-analysis of number of total cells, β-cells, and proliferating β- and non-β-cells per islet MT. In parallel, insulin secretion, intracellular insulin and ATP contents, and Caspase 3/7 activity were analyzed to obtain a comprehensive overview of islet MT function and viability. We observed that 4-day harmine treatment increased β- and non-β-cell proliferation, NKX6.1 expression, and basal and stimulated insulin secretion in a dose-dependent manner, while fold-stimulation of secretion peaked at intermediate harmine doses. Interestingly, 15-day harmine treatment led to a general reduction in harmine's proliferative effects as well as altered dose-dependent trends. The described methodology provides a unique tool for high-throughput evaluation of short- and long-term changes in human β-cell proliferation, count and fraction along with a variety of functional parameters, in a representative 3D human islet model.
通过诱导增殖来恢复β细胞数量代表了治疗糖尿病的一种有吸引力的治疗方法。然而，完整分散的原代胰岛会迅速降低活力和功能，这对它们在增殖研究中的实验应用构成了重大挑战。在这里，我们描述了一种使用重新聚集的原代人胰岛或胰岛微组织 (MT) 评估化合物对β细胞增殖和计数的影响的新方法，这些 MT 显示出均匀的大小和组织结构，以及在培养 4 周内具有稳健和稳定的功能和活力。我们利用该平台评估了 harmine 对β细胞增殖和功能的短期和长期剂量依赖性影响。在化合物处理和 EdU 掺入后，对胰岛 MT 进行染色和共聚焦成像，以检测 DAPI（核标记物）、NKX6.1（β细胞标记物）和 EdU（增殖标记物），从而可以自动分析每个胰岛 MT 的总细胞、β细胞和增殖的β和非β细胞的数量。同时，分析胰岛素分泌、细胞内胰岛素和 ATP 含量以及 Caspase 3/7 活性，以全面了解胰岛 MT 的功能和活力。我们观察到，4 天 harmine 处理以剂量依赖性方式增加了β和非β细胞的增殖、NKX6.1 表达以及基础和刺激胰岛素分泌，而分泌的倍数刺激在中等 harmine 剂量下达到峰值。有趣的是，15 天 harmine 处理导致 harmine 的增殖作用普遍降低，并且剂量依赖性趋势发生改变。所描述的方法为在代表性的 3D 人胰岛模型中，对人β细胞增殖、计数和分数以及各种功能参数的短期和长期变化进行高通量评估提供了独特的工具。
