High content screening miniaturization and single cell imaging of mature human feeder layer-free iPSC-derived neurons.
High content screening miniaturization and single cell imaging of mature human feeder layer-free iPSC-derived neurons.
复制标题
DOI：
10.1016/j.slasd.2022.10.002
复制
发表时间：
2023-09
期刊：
影响因子：
3.1
通讯作者：
Lazo, John S.
中科院分区：
文献类型：
--
作者：
Sharlow, Elizabeth R.;Llaneza, Danielle C.;Grever, William E.;Mingledorff, Garnett A.;Mendelson, Anna J.;Bloom, George S.;Lazo, John S.
人诱导多能干细胞（iPSC）分化而来的神经元正越来越多地用于高内涵成像和筛选。然而，iPSC向神经元的分化和成熟过程耗时较长，通常需要8周以上。遗憾的是，处于分化和成熟阶段的iPSC衍生神经元培养物还容易迁移并聚集成神经节样簇，这给单细胞分析带来了挑战，尤其是在微型化培养模式中。我们利用特定的细胞外基质和低氧培养条件来促进人皮层神经元的分化和成熟，在此基础上，进一步调整了神经元祖细胞在微型化（如96孔板）培养中的接种密度以及无饲养层培养条件，以减少神经元聚集，增强单细胞识别能力，并降低长时间神经元细胞培养后常见的边缘效应。随后开发的算法进一步提升了从大细胞聚集体中区分和识别单个成熟神经元（以NeuN表达为标识）的能力，大细胞聚集体会被排除在图像分析之外。在分化和成熟阶段加入星形胶质细胞条件培养基，显著提高了分化4周后检测到的成熟神经元（即NeuN阳性）的比例（从约10%提高到约30%）。使用这种优化检测系统进行的初步概念验证研究表明，在基于群体以及基于图像的神经毒性检测模式中，边缘效应可忽略不计，且Z因子表现良好。此外，莫昔克丁，一种经美国食品药品监督管理局（FDA）批准且有神经毒性不良反应记录的药物，在这两种筛选模式中均被识别为阳性结果。这种微型化、无饲养层的培养模式以及图像分析算法提供了一个基础成像和筛选平台，能够对分化的人类神经元进行定量单细胞分析。
Human induced pluripotent stem cell (iPSC)-derived neurons are being increasingly used for high content imaging and screening. However, iPSC-derived neuronal differentiation and maturation is time-intensive, often requiring >8 weeks. Unfortunately, the differentiating and maturing iPSC-derived neuronal cultures also tend to migrate and coalesce into ganglion-like clusters making single-cell analysis challenging, especially in miniaturized formats. Using our defined extracellular matrix and low oxygen culturing conditions for the differentiation and maturation of human cortical neurons, we further modified neuronal progenitor cell seeding densities and feeder layer-free culturing conditions in miniaturized formats (i.e., 96 well) to decrease neuronal clustering, enhance single-cell identification and reduce edge effects usually observed after extended neuronal cell culture. Subsequent algorithm development refined capabilities to distinguish and identify single mature neurons, as identified by NeuN expression, from large cellular aggregates, which were excluded from image analysis. Incorporation of astrocyte conditioned medium during differentiation and maturation periods significantly increased the percentage (i.e., ~10% to ~30%) of mature neurons (i.e., NeuN+) detected at 4-weeks post-differentiation. Pilot, proof of concept studies using this optimized assay system yielded negligible edge effects and robust Z-factors in population-based as well as image-based neurotoxicity assay formats. Moreover, moxidectin, an FDA-approved drug with documented neurotoxic adverse effects, was identified as a hit using both screening formats. This miniaturized, feeder layer-free format and image analysis algorithm provides a foundational imaging and screening platform, which enables quantitative single-cell analysis of differentiated human neurons.
登录
查看更多内容
影响因子：
64.5
作者：
Mariani J;Coppola G;Zhang P;Abyzov A;Provini L;Tomasini L;Amenduni M;Szekely A;Palejev D;Wilson M;Gerstein M;Grigorenko EL;Chawarska K;Pelphrey KA;Howe JR;Vaccarino FM
通讯作者：
Vaccarino FM
DOI：
10.1177/24725552211000671
发表时间：
2021-07
期刊：
SLAS discovery : advancing life sciences R & D
影响因子：
--
作者：
MacMullen C;Davis RL
通讯作者：
Davis RL
影响因子：
9.5
作者：
Koroleva, Anastasia;Deiwick, Andrea;Chichkov, Boris
通讯作者：
Chichkov, Boris
影响因子：
1.2
作者：
Chacon, Jose;Rogers, Crystal D.
通讯作者：
Rogers, Crystal D.
DOI：
10.1002/trc2.12179
发表时间：
2021
期刊：
Alzheimer's & dementia (New York, N. Y.)
影响因子：
--
作者：
Cummings J;Lee G;Zhong K;Fonseca J;Taghva K
通讯作者：
Taghva K