Embryoid body formation from ESC/iPSC
Materials Required
Principle
Embryoid bodies are three-dimensional aggregates formed from ESCs or iPSCs in non-adherent suspension conditions, and EB formation is used to initiate spontaneous differentiation and assess differentiation potential across ectodermal, mesodermal, and endodermal lineages[1][2][9]. EB readouts include aggregate formation, EB size and morphology, lineage-marker expression by immunostaining or qPCR, and downstream lineage differentiation outcomes[1][9][10][12].
MCE has not independently verified the accuracy of these methods. They are for reference only.
Experimental Materials
• Use pluripotent ESCs or iPSCs as the starting cell population; use EB differentiation medium reported in the selected protocol, such as serum-free APEL for spin EB differentiation or E8/PVA-based chemically defined medium for human PSC EB formation[5][8].
• Use non-adherent culture conditions, low-attachment plates, bacterial-grade dishes, hanging drops, V-bottom 96-well plates, AggreWell plates, or cell-repellent microwells depending on the EB-format selected[2][3][4][6][7][13].
• Use lineage-marker assays only when matched to the study goal; published EB pluripotency assessment used markers of ectoderm, mesoderm, and endoderm by immunostaining and gene-expression analysis[1][9].
• Hematopoietic EB studies used hematopoietic differentiation readouts after defined EB formation, while cardiac EB studies used cardiomyocyte differentiation readouts after controlled EB-size formation[4][5][11][12].
• Use a humidified cell-culture incubator, sterile cell-culture workspace, microscope, centrifuge for spin-EB formats, low-attachment or non-adherent cultureware, V-bottom 96-well plates, AggreWell plates, or microfabricated cell-repellent microwell arrays as required by the selected EB method[3][4][5][7][13].
• Use non-adherent culture conditions, low-attachment plates, bacterial-grade dishes, hanging drops, V-bottom 96-well plates, AggreWell plates, or cell-repellent microwells depending on the EB-format selected[2][3][4][6][7][13].
• Use lineage-marker assays only when matched to the study goal; published EB pluripotency assessment used markers of ectoderm, mesoderm, and endoderm by immunostaining and gene-expression analysis[1][9].
• Hematopoietic EB studies used hematopoietic differentiation readouts after defined EB formation, while cardiac EB studies used cardiomyocyte differentiation readouts after controlled EB-size formation[4][5][11][12].
• Use a humidified cell-culture incubator, sterile cell-culture workspace, microscope, centrifuge for spin-EB formats, low-attachment or non-adherent cultureware, V-bottom 96-well plates, AggreWell plates, or microfabricated cell-repellent microwell arrays as required by the selected EB method[3][4][5][7][13].
Experimental Procedure
• Begin with undifferentiated ESCs or iPSCs and choose one EB-format before seeding, because suspension culture, methylcellulose or semisolid culture, hanging-drop culture, forced aggregation, AggreWell, and microwell systems produce different EB size distributions and differentiation outputs[2][3][4][7][10][11][12][13].
• For protocols prioritizing uniformity, seed defined cell numbers into low-attachment round-bottom 96-well plates and aggregate by centrifugation, or seed dissociated cells into AggreWell or cell-repellent microwells to control aggregate size[4][5][7][13].
• For classical EB formation, culture ESC/iPSC clumps or dissociated cells under non-adherent suspension conditions, in hanging drops, or in methylcellulose/semisolid medium, because these are reported EB-induction formats[2][3].
• For spin EB formation, place known numbers of hESCs into low-attachment round-bottom 96-well plates in serum-free medium and centrifuge to initiate uniform EB formation[4][5].
• For AggreWell or microwell formation, seed cells into microwell-based non-adherent arrays to generate many EBs with more controlled size and shape than conventional suspension methods[7][11][13].
• Record EB number, morphology, and size distribution, because EB size and aggregate heterogeneity influence differentiation trajectory and lineage output[10][11][12].
• Assess differentiation by comparing lineage-marker expression for ectoderm, mesoderm, and endoderm using immunostaining or qPCR, and include an undifferentiated PSC control and, when available, a reference ESC or validated iPSC line for comparison[1][9].
• Interpret EB formation as an in vitro differentiation-potential assay rather than proof of full developmental equivalence, because EB assays assess spontaneous or directed lineage differentiation under culture conditions[1][9].
• For protocols prioritizing uniformity, seed defined cell numbers into low-attachment round-bottom 96-well plates and aggregate by centrifugation, or seed dissociated cells into AggreWell or cell-repellent microwells to control aggregate size[4][5][7][13].
• For classical EB formation, culture ESC/iPSC clumps or dissociated cells under non-adherent suspension conditions, in hanging drops, or in methylcellulose/semisolid medium, because these are reported EB-induction formats[2][3].
• For spin EB formation, place known numbers of hESCs into low-attachment round-bottom 96-well plates in serum-free medium and centrifuge to initiate uniform EB formation[4][5].
• For AggreWell or microwell formation, seed cells into microwell-based non-adherent arrays to generate many EBs with more controlled size and shape than conventional suspension methods[7][11][13].
• Record EB number, morphology, and size distribution, because EB size and aggregate heterogeneity influence differentiation trajectory and lineage output[10][11][12].
• Assess differentiation by comparing lineage-marker expression for ectoderm, mesoderm, and endoderm using immunostaining or qPCR, and include an undifferentiated PSC control and, when available, a reference ESC or validated iPSC line for comparison[1][9].
• Interpret EB formation as an in vitro differentiation-potential assay rather than proof of full developmental equivalence, because EB assays assess spontaneous or directed lineage differentiation under culture conditions[1][9].
Troubleshooting
Problem: EB size is heterogeneous.
• Possible Cause: Conventional suspension methods and variable starting aggregate size produce heterogeneous EB populations.• Literature-supported Solution: Use defined-cell-number forced aggregation, V-bottom plates, AggreWell plates, or microfabricated cell-repellent microwells when uniform EB size is required[4][6][7][11][13].
Problem: Differentiation output varies between experiments or cell lines.
• Possible Cause: EB size, colony or aggregate size heterogeneity, EB method, and intrinsic line-to-line differences can alter differentiation trajectories.• Literature-supported Solution: Standardize input cell number, EB formation format, and EB size range, and compare lines under the same EB conditions[6][10][11][12].
Problem: EB assay does not clearly demonstrate three-germ-layer differentiation.
• Possible Cause: Aggregate formation alone does not verify lineage differentiation.• Literature-supported Solution: Add immunostaining or gene-expression analysis for ectodermal, mesodermal, and endodermal markers after EB differentiation[1][9].
References:
- [1]. Itskovitz-Eldor J, et al. Differentiation of human embryonic stem cells into embryoid bodies comprising the three embryonic germ layers. Mol Med. 2000;6(2):88-95. [Content Brief]
- [2]. Kurosawa H. Methods for inducing embryoid body formation: in vitro differentiation system of embryonic stem cells. J Biosci Bioeng. 2007;103(5):389-398. [Content Brief]
- [3]. Cerdan C, et al. Formation and hematopoietic differentiation of human embryoid bodies by suspension and hanging drop cultures. Curr Protoc Stem Cell Biol. 2007;Chapter 1:Unit 1D.2. [Content Brief]
- [4]. Ng ES, et al. Forced aggregation of defined numbers of human embryonic stem cells into embryoid bodies fosters robust, reproducible hematopoietic differentiation. Blood. 2005;106(5):1601-1603. [Content Brief]
- [5]. Ng ES, et al. A protocol describing the use of a recombinant protein-based, animal product-free medium (APEL) for human embryonic stem cell differentiation as spin embryoid bodies. Nat Protoc. 2008;3(5):768-776. [Content Brief]
- [6]. Burridge PW, et al. Improved human embryonic stem cell embryoid body homogeneity and cardiomyocyte differentiation from a novel V-96 plate aggregation system highlights interline variability. Stem Cells. 2007;25(4):929-938. [Content Brief]
- [7]. Antonchuk J. Formation of embryoid bodies from human pluripotent stem cells using AggreWell plates. Methods Mol Biol. 2013;946:523-533. [Content Brief]
- [8]. Lin Y. Embryoid body formation from human pluripotent stem cells in chemically defined E8 media. StemBook. 2014. [Content Brief]
- [9]. Sheridan SD, et al. Analysis of embryoid bodies derived from human induced pluripotent stem cells as a means to assess pluripotency. Stem Cells Int. 2012;2012:738910. [Content Brief]
- [10]. Bauwens CL, et al. Control of human embryonic stem cell colony and aggregate size heterogeneity influences differentiation trajectories. Stem Cells. 2008;26(9):2300-2310. [Content Brief]
- [11]. Mohr JC, et al. The microwell control of embryoid body size in order to regulate cardiac differentiation of human embryonic stem cells. Biomaterials. 2010;31(7):1885-1893. [Content Brief]
- [12]. Hong SH, et al. Multiparameter comparisons of embryoid body differentiation toward human stem cell applications. Stem Cell Res. 2010;5(2):120-130. [Content Brief]
- [13]. Pettinato G, et al. Formation of well-defined embryoid bodies from dissociated human induced pluripotent stem cells using microfabricated cell-repellent microwell arrays. Sci Rep. 2014;4:7402. [Content Brief]
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