Embryoid Body (EB) size a determining factor for differentiation efficiencies

Note: This is a review of the published article listed below. All information, quotes, figures, methods, and findings mentioned in this review are from that article, and are the property of its authors and/or the publication in which the article originally appeared.

Original Research Paper

Hydrophobic surfaces for enhanced differentiation of embryonic stem cell-derived embryoid bodies

Bahram Valamehr, Steven J. Jonas, Julien Polleux, Rong Qiao, Shuling Guo, Eric H. Gschweng, Bangyan Stiles, Korey Kam, Tzy-Jiun M. Luo, Owen N. Witte, Xin Liu, Bruce Dunn, and Hong Wu

PNAS, September 23, 2008, vol. 105, no. 38, 14459–14464
http://www.pnas.org/content/105/38/14459.full

Review

The formation of EB's is a common and critical intermediate during the in vitro differentiation of human embryonic and induced pluripotent stem cells (hESCs, hiPSCs) into specific cell types. The lineage differentiation programs within the EB closely resemble that of a developing embryo in vivo. EBs are typically generated by removing stem cell colonies after 5-6 days of proliferation from the feeder/matrix contact using collagenase. Removed colonies are then grown in suspension culture on low attachment plates in differentiation medium. EBs are formed within a few days of suspension culture after which various differentiation strategies are employed. Cultures maintained by enzymatic passage contain variable sized stem cell colonies and result in the formation of a heterogeneous EB population, varying in size and morphology.

This study by Bahram Valamehr et. al., (2007) provides evidence that that EB size can determine cellular viability, proliferation, and differentiation potential in mice (Figure 1, 5) and in humans (Figure 3, 5.) They demonstrate that intermediate sized EBs (100-300um in diameter) generated using hydrophobic substrates were the most proliferative, held the greatest differentiation potential and had the lowest rate of cell death. This study highlights the importance of consistent and controlled EB culture management which may significantly improve the efficiency of ES cell differentiation.

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  Fig 1. EB size influences cellular viability, proliferation, and differentiation potential. (A) Day 4 EBs were manually separated into three size categories: small, intermediate, and large. (Scale bars, 250 um.) (B) EBs of the intermediate size contain the lowest number of apoptotic cells. n = 3. (C) qRT-PCR analysis of lineage marker expression for ectoderm (Nestin and Tau,) mesoderm
  [Brachyury (Bry) and Flk1,] and endoderm (Pdx1 and Sox17) in each subpopulation. The level of 18S serves as an internal control. n = 3. Data presented as mean +SD. **, P<0.01.

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  Fig. 2. Uniform human EB formation on hydrophobic surfaces. (A) Comparison of EBs derived from HSF1 human ES cells cultured either on LAC plates (Upper) or on C18 surfaces (Lower) and maintained in continuous culture for 8 days. (Scale bars, 250um.) (B) At day 7 of culture, EBs cultured on C18 display lower apoptosis profile as analyzed by TUNEL staining. n =5. (C) A higher percentage of cells derived from EBs cultured on C18 resides in S/G2/M after 7 days of culture. n =5. Data presented as mean +SD; **, P <0.01.

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  Fig. 3. Serum-free differentiation is enhanced by PDMS into the three somatic lineages. (A) Mouse and human ES cells were directed toward neuronal lineage for a total of 14 and 18 days, respectively, and scored for number of Tuj1 (neuronal marker)-positive cells under serum-free conditions. (B Left) Mouse and human ES cells were directed toward hematopoietic lineage for a total of 12 and 20 days, respectively, and scored for the appearance of various colonies in methylcellulose assay. (Right Top) Representative images of macrophage-containing colonies under brightfield microscopy for mouse and human. (Right Middle) The existence of macrophage colonies was confirmed by cytospin for mouse and human. (Right Bottom) Erythroid-containing colonies were confirmed by DAB staining for mouse and human. (Scale bars, 100 µm.) (C) Mouse and human ES cells were directed toward endoderm lineage for a total of 12 and 16 days, respectively, and assayed for associated gene expression. n = 6. Data presented as mean ± SD; all data, P = 0.05.