The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, USA.
School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA, USA.
Biomed Microdevices. 2014 Feb;16(1):79-90. doi: 10.1007/s10544-013-9807-3.
The differentiation of pluripotent stem cells as embryoid bodies (EBs) remains a common method for inducing differentiation toward many lineages. However, differentiation via EBs typically yields a significant amount of heterogeneity in the cell population, as most cells differentiate simultaneously toward different lineages, while others remain undifferentiated. Moreover, physical parameters, such as the size of EBs, can modulate the heterogeneity of differentiated phenotypes due to the establishment of nutrient and oxygen gradients. One of the challenges in examining the cellular composition of EBs is the lack of analytical methods that are capable of determining the phenotype of all of the individual cells that comprise a single EB. Therefore, the objective of this work was to examine the ability of a microfluidic cell trapping array to analyze the heterogeneity of cells comprising EBs during the course of early differentiation. The heterogeneity of single cell phenotype on the basis of protein expression of the pluripotent transcription factor OCT-4 was examined for populations of EBs and single EBs of different sizes at distinct stages of differentiation. Results from the cell trap device were compared with flow cytometry and whole mount immunostaining. Additionally, single cells from dissociated pooled EBs or individual EBs were examined separately to discern potential differences in the value or variance of expression between the different methods of analysis. Overall, the analytical method described represents a novel approach for evaluating how heterogeneity is manifested in EB cultures and may be used in the future to assess the kinetics and patterns of differentiation in addition to the loss of pluripotency.
多能干细胞向胚状体 (EB) 的分化仍然是诱导向许多谱系分化的常用方法。然而，通过 EB 进行分化通常会导致细胞群体中存在大量异质性，因为大多数细胞同时向不同谱系分化，而其他细胞则保持未分化状态。此外，由于营养物质和氧气梯度的建立，EB 的大小等物理参数可以调节分化表型的异质性。检查 EB 中细胞组成的一个挑战是缺乏能够确定单个 EB 中所有单个细胞表型的分析方法。因此，这项工作的目的是检查微流控细胞捕获阵列在早期分化过程中分析 EB 中细胞异质性的能力。基于多能转录因子 OCT-4 的蛋白质表达，检查了不同大小的 EB 群体和单个 EB 在不同分化阶段的单细胞表型异质性。细胞捕获装置的结果与流式细胞术和全胚免疫染色进行了比较。此外，还分别检查了来自分离的汇集 EB 或单个 EB 的单个细胞，以辨别不同分析方法之间表达值或方差的潜在差异。总体而言，所描述的分析方法代表了一种评估 EB 培养物中异质性表现方式的新方法，并且将来可能用于评估分化的动力学和模式以及多能性的丧失。
