Barry Christopher, Schmitz Matthew T, Propson Nicholas E, Hou Zhonggang, Zhang Jue, Nguyen Bao K, Bolin Jennifer M, Jiang Peng, McIntosh Brian E, Probasco Mitchell D, Swanson Scott, Stewart Ron, Thomson James A, Schwartz Michael P, Murphy William L
1 Morgridge Institute for Research, Madison, WI 53705, USA.
2 Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109, USA (current address).
Exp Biol Med (Maywood). 2017 Nov;242(17):1679-1689. doi: 10.1177/1535370217715028. Epub 2017 Jun 9.
The aim of the present study was to test sample reproducibility for model neural tissues formed on synthetic hydrogels. Human embryonic stem (ES) cell-derived precursor cells were cultured on synthetic poly(ethylene glycol) (PEG) hydrogels to promote differentiation and self-organization into model neural tissue constructs. Neural progenitor, vascular, and microglial precursor cells were combined on PEG hydrogels to mimic developmental timing, which produced multicomponent neural constructs with 3D neuronal and glial organization, organized vascular networks, and microglia with ramified morphologies. Spearman's rank correlation analysis of global gene expression profiles and a comparison of coefficient of variation for expressed genes demonstrated that replicate neural constructs were highly uniform to at least day 21 for samples from independent experiments. We also demonstrate that model neural tissues formed on PEG hydrogels using a simplified neural differentiation protocol correlated more strongly to in vivo brain development than samples cultured on tissue culture polystyrene surfaces alone. These results provide a proof-of-concept demonstration that 3D cellular models that mimic aspects of human brain development can be produced from human pluripotent stem cells with high sample uniformity between experiments by using standard culture techniques, cryopreserved cell stocks, and a synthetic extracellular matrix. Impact statement Pluripotent stem (PS) cells have been characterized by an inherent ability to self-organize into 3D "organoids" resembling stomach, intestine, liver, kidney, and brain tissues, offering a potentially powerful tool for modeling human development and disease. However, organoid formation must be quantitatively reproducible for applications such as drug and toxicity screening. Here, we report a strategy to produce uniform neural tissue constructs with reproducible global gene expression profiles for replicate samples from multiple experiments.
本研究的目的是测试在合成水凝胶上形成的模型神经组织的样本可重复性。将人胚胎干（ES）细胞来源的前体细胞培养在合成聚乙二醇（PEG）水凝胶上，以促进其分化并自组织形成模型神经组织构建体。将神经祖细胞、血管细胞和小胶质前体细胞组合在PEG水凝胶上以模拟发育时间，从而产生具有三维神经元和神经胶质组织、有组织的血管网络以及具有分支形态的小胶质细胞的多组分神经构建体。对全局基因表达谱进行Spearman等级相关分析以及对表达基因的变异系数进行比较，结果表明，来自独立实验的样本的重复神经构建体在至少第21天之前高度一致。我们还证明，使用简化的神经分化方案在PEG水凝胶上形成的模型神经组织与单独在组织培养聚苯乙烯表面上培养的样本相比，与体内脑发育的相关性更强。这些结果提供了一个概念验证，即通过使用标准培养技术、冷冻保存的细胞库和合成细胞外基质，可以从人多能干细胞产生模拟人类脑发育方面的三维细胞模型，且实验之间的样本具有高度一致性。影响声明多能干细胞（PS）的特征在于具有自组织成类似胃、肠、肝、肾和脑组织的三维“类器官”的内在能力，为模拟人类发育和疾病提供了一个潜在的强大工具。然而，类器官的形成对于药物和毒性筛选等应用必须在数量上具有可重复性。在此，我们报告了一种策略，可为来自多个实验的重复样本生成具有可重复的全局基因表达谱的均匀神经组织构建体。
