Embryoid Body (EB) Formation

An important alternative method to the teratoma assay is the in vitro approach involving the generation of embryoid bodies (EBs) from hiPSCs. EBs are three-dimensional aggregation of three germ layer cells. The formation of EBs is a routine method for the differentiation of hiPSCs into different cell lineages. One of the major advantages of this method is that it is performed in vitro with standard tissue culture approaches and materials, thereby avoiding the regulatory problems and substantial costs associated with maintaining immunedeficient mice. EBs can be easily grown in a suspension culture dish in the laboratory and can be scaled up without much difficulty once the appropriate conditions are established. Unlike the teratoma assay where hiPSCs fail to form teratomas for unknown reasons, hiPSCs can readily form EBs by a variety of ways, allowing for trilineage differentiation and analysis in a more controlled, reproducible manner.

Figure 1. hiPSCs are pluripotent.

Materials and Reagents

EDTA/PBS solution
Dilute 0.5 mL 0.5 M EDTA (pH 8.0) in 500 mL PBS (calcium and magnesium free), add 0.9 g NaCl, mix and filtrate. The final EDTA concentration is 0.5 mM.
Matrigel coated plate/dish
Use 1.5 mL of DMEM/F12 to resuspend 4 mg frozen Matrigel. Mix the Matrigel with the media well, plate 6 mL in one six-well plate (1 mL per well) or 6 mL in one 10 cm dish and shake well to cover the entire surface. Leave the plates/dishes at room temperature for at least 30 min or at 4°C overnight.
Vitronectin coated plate/dish
Apply the same procedure of Matrigel coating to coat vitronectin.
E8/Essential 8 media
The media contain DMEM/F12, L-ascorbic acid-2-phosphate magnesium (64 mg/L), sodium selenium (14 µg/L), FGF2 (100 µg/L), insulin (19.4 mg/L), NaHCO₃ (543 mg/L) and transferrin (10.7 mg/L), TGFβ1 (2 µg/L) or NODAL (100 µg/L). Osmolarity of all media was adjusted to 340 mOsm at pH7.4.
E6/Essential 6 medium
E6 is similar to E8 medium but without FGF2 and TGF-β.
PVA containing media
Polyvinyl Alcohol (PVA) powder is added into specific medium (E8 or E6) at 4 mg/mL, mixed and stirred for 1 h at room temperature, filtered and then stored at 4°C.
Poly-HEMA coated dish
Poly-HEMA solution is made at 20 mg/mL by dissolve 1 g Poly-HEMA in 50 mL 95% ethanol on rock overnight in 37°C. To coat the dishes, pour the Poly-HEMA/ethanol mixture to cover the dishes and let it dry out overnight at room temperature.

Assay Procedure

hPSCs are cultured in E8 medium on matrigel or vitronectin coated plates, and medium is changed daily.
Two days before EB formation, 60-70% confluent hPSCs are passaged onto matrigel or vitronectin coated plates/dishes with EDTA/PBS. The passage ratio is usually 1:3 or 1:4, in E8 media with 10 μM ROCK inhibitor to increase cell survival.
The next day, change media to E8 medium without ROCK inhibitor.
On the day of EB formation when the cells grow to 60-80% confluence, cells are washed once and then incubated in EDTA/PBS for 3-15 min to dissociate colonies to cell clumps or single cells according to EB formation methods.
1) Incubation for 3-5 min - Self-aggregated EB formation.
2) Incubation for 10-15min - Forced aggregation by hanging drop or using Microwell.
Two methods are used to harvest the cells according to EDTA treatment time.
1) 3-5 minutes EDTA incubation where most cells are still attached to plate surface. Aspirate EDTA/PBS, wash cell clumps off the plate by E8/PVA (5 mL/dish) with ROCK inhibitor. This method is suitable for Self-aggregated EB formation.
2) 10-15 minutes EDTA incubation where most cells detach from the plate surface to become single cells or small aggregates. Gently break the aggregates with PBS/EDTA (5 mL/dish) by pipetting, and then transfer the cells into 15 mL tube, neutralize with equal volume of E8/PVA medium with ROCK inhibitor, count cell number, and spin the cells down at 1,000 rpm for 5 min. Finally, resuspend the cells in E8/PVA medium with ROCK inhibitor. This method is suitable for forced aggregation, such as hanging drop or using Microwell.
To form self-aggregated EB, suspend cell clumps into low attachment dishes or poly-HEMA coated dishes via 1:1 passage to allow self-aggregation in 37°C incubator overnight. The next day, EBs should form in various sizes.
To form hanging drop EBs, single cell drops (2000 cells/20 µL) are hanging cultured on lid of the dishes. Incubate in 37°C incubator overnight. On the second day, EBs should form with uniform size. The aggregated EBs can be washed off into low attachment dishes or poly-HEMA coated dishes.
To form EBs in Microwells, rinse each well by DMEM/F12 before use. Add 0.5 mL E8/PVA medium to each well that will be used and centrifuge to remove air bubbles. Add 1.5 x 10⁶ cells/1.5 mL into each well to generate the desired size of EBs. Centrifuge the Microwell plate at 100 g, 3 min to capture cells in the wells. Incubate the plate in 37°C incubator overnight. On the second day EB should form with uniform size. Gently pipet the medium up and down in Microwell to remove the EBs and transfer them into low attached dishes or poly-HEMA coated dishes.

After EBs are formed and transferred to low attachment dishes or poly-HEMA coated dishes, tilt the plate at 30°-45° angle to allow the EBs to gather at the bottle of well. Gently remove most medium with pipette, and change media to desired differentiation conditions for specific cell type differentiation. For example, in spontaneous differentiation, the medium is switched to E6 medium to culture 9 to 14 days. The medium can be changed every 2 days.

Figure 2. Methods to form embryoid body.

Troubleshooting

For EDTA dissociation, different lines may have various processing times.
Self-aggregation forms heterogeneous size and irregularly shape EBs, while hanging drops or using Microwell form uniform size and shape EBs.
In order to improve cell survival and EB formation, the cells need to be freshly passaged. We recommend passing cells (1:3 or 1:4) 2 days before EB formation.
ROCK inhibitor can be used to increase cell survival while promoting cell attachment to the surface of uncoated plates.
To promote EB formation under self-aggregation, the cell dissociation time by EDTA should be strictly monitored to avoid single cell dissociation. Aggregation of cell clumps increases the formation of EB.
It is strongly recommended to add PVA to help EB formation, but PVA can be removed from media after EBs were formed.
Differentiation medium could be directly used in EB formation instead of E8 medium.

References

Kurosawa H. et al. Methods for inducing embryoid body formation: in vitro differentiation system of embryonic stem cells. J Biosci Bioeng, 2007, 103(5): 389-398.
Lin Y. et al. Embryoid body formation from human pluripotent stem cells in chemically defined E8 media. StemBook, 2014.

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