Outten Joel T, Cheng Xin, Gadue Paul, French Deborah L, Diamond Scott L
Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, PA 19104, USA.
Stem Cell Res. 2011 Mar;6(2):129-42. doi: 10.1016/j.scr.2010.11.001. Epub 2010 Nov 16.
Serum-free differentiation protocols of human embryonic stem cells (hESCs) offer the ability to maximize reproducibility and to develop clinically applicable therapies. We developed a high-throughput, 96-well plate, four-color flow cytometry-based assay to optimize differentiation media cocktails and to screen a variety of conditions. We were able to differentiate hESCs to all three primary germ layers, screen for the effect of a range of activin A, BMP4, and VEGF concentrations on endoderm and mesoderm differentiation, and perform RNA-interference (RNAi)-mediated knockdown of a reporter gene during differentiation. Cells were seeded in suspension culture and embryoid bodies were induced to differentiate to the three primary germ layers for 6 days. Endoderm (CXCR4(+)KDR(-)), mesoderm (KDR(+)SSEA-3(-)), and ectoderm (SSEA-3(+)NCAM(+)) differentiation yields for H9 cells were 80 ± 11, 78 ± 7, and 41 ± 9%, respectively. Germ layer identities were confirmed by quantitative PCR. Activin A, BMP4, and bFGF drove differentiation, with increasing concentrations of activin A inducing higher endoderm yields and increasing BMP4 inducing higher mesoderm yields. VEGF drove lateral mesoderm differentiation. RNAi-mediated knockdown of constitutively expressed red fluorescent protein did not affect endoderm differentiation. This assay facilitates the development of serum-free protocols for hESC differentiation to target lineages and creates a platform for screening small molecules or RNAi during ESC differentiation.
人胚胎干细胞（hESCs）的无血清分化方案能够实现最大化的可重复性，并开发出临床适用的疗法。我们开发了一种基于96孔板、四色流式细胞术的高通量检测方法，用于优化分化培养基组合并筛选各种条件。我们能够将hESCs分化为所有三个原始胚层，筛选一系列激活素A、骨形态发生蛋白4（BMP4）和血管内皮生长因子（VEGF）浓度对内胚层和中胚层分化的影响，并在分化过程中进行RNA干扰（RNAi）介导的报告基因敲低。将细胞接种于悬浮培养中，诱导胚状体向三个原始胚层分化6天。H9细胞的内胚层（CXCR4(+)KDR(-)）、中胚层（KDR(+)SSEA-3(-)）和外胚层（SSEA-3(+)NCAM(+)）分化率分别为80±11%、78±7%和41±9%。通过定量PCR确认胚层身份。激活素A、BMP4和碱性成纤维细胞生长因子（bFGF）驱动分化，激活素A浓度增加诱导更高的内胚层产量，BMP4浓度增加诱导更高的中胚层产量。VEGF驱动侧中胚层分化。RNAi介导的组成型表达红色荧光蛋白的敲低不影响内胚层分化。该检测方法有助于开发hESCs分化为目标谱系的无血清方案，并为ESC分化过程中筛选小分子或RNAi创造了一个平台。
