Embryoid Body (EB) Formation from Human iPSCs
and Mouse ESCs

Embryoid bodies (EBs) are floating spherical aggregates of human iPSCs or mouse ESCs used to assess pluripotency by testing their ability to differentiate. To form EBs, cells are passaged as usual but plated into non tissue culture–treated dishes to prevent attachment, using EB medium without bFGF. The following protocols outline iPSC and mESC differentiation using complete KnockOut Serum Replacement EB medium.

To form EBs from hiPSCs, cells are passaged as usual but plated into non tissue culture–treated dishes to prevent attachment, using EB medium without bFGF. The following protocols outline iPSC differentiation using complete KnockOut Serum Replacement EB medium.

Prior to starting, ensure that any media is equilibrated to 37°C and appropriately gassed.

Preparing complete KSR EB Medium

1. To prepare 100 mL of Complete KnockOut Serum Replacement EB medium, aseptically combine the components listed in the table below.
   
   

   Component	Stock concentration	Final concentration	Volume
   Knockout DMEM/F12	-	1X	78 mL
   GlutaMAX Supplement	200 mM	2 mM	1 mL
   KnockOut Serum Replacement	-	20%	20 mL
   MEM Non-Essential Amino Acids Solution (100X)	10 mM	0.1 mM	1

2. Just before pre-equilibrating the complete medium to temperature and gases, aseptically add the required volume of β-mercaptoethanol (55 mM stock concentration) for a 0.1 mM final concentration. For example, to prepare 100 mL of KSR-FF medium add 182 μL of 55 mM β-mercaptoethanol (1:550 dilution) Alternatively, the β-mercaptoethanol may be added to the 1X completed medium and stored at 2–8°C for up to one week.
3. Aliquot appropriate volume (Table 1) of KSR EB Medium needed for the day to a centrifuge tube warm to 37°C.

Forming embryoid bodies from iPSCs

1. Observe the human iPSCs growing in complete KSR-Feeder Free medium under the microscope to confirm that the cells are 70–80% confluent and ready to be subcultured.

2. Cut out and remove any differentiated iPSC or hESC colonies prior to passaging the culture.
3. Pre-warm the required volume of Dispase in a 37 °C water bath. Refer to Table 1 below for details on the volumes required.
4. Pre-equilibrate the required volume of KSR-EB medium in a 37°C water bath for 15 min. Refer to Table 1 below for details on the volumes required.
5. Aspirate the spent medium from the culture dish using a pipette and rinse the cells twice with D-PBS.
6. Gently add pre-warmed Dispase solution to the culture dish (e.g., 1 mL of Dispase solution per 60-mm culture dish). Swirl the culture dish to coat the entire cell surface.
7. Incubate the culture dish at 37°C for 3 minutes.
8. Remove the dish from the incubator, aspirate the Dispase solution, and gently wash the cells with DPBS.
9. Gently scrape the cells off the surface of the culture dish using a cell scraper and transfer the cells to a sterile 15 mL centrifuge tube.
10. Rinse the culture dish twice with DPBS, gently “spraying off” any cells that have not detached. Pool the rinse with the cells in the 15 mL tube.
11. Centrifuge the tube at 200 × g for 5 minutes at room temperature to pellet the cells.
12. Carefully aspirate the supernatant without disturbing the cell pellet and discard it.
13. Gently flick the tube to fully dislodge the cell pellet from the tube bottom.
14. Gently resuspend the cells in pre-equilibrated complete KSR-EB medium using a 5 mL serological pipette. Do not triturate. 

15. One 60 mm dish of cells can be transferred to one 60 mm EB dish or one 100 mm EB dish. Place the cells onto a 60 mm or a 100 mm non tissue culture treated dish.
16. Place the culture dish in a 37°C incubator with a humidified atmosphere of 4 to 6% CO₂ in air.
17. Change the medium on the EBs every other day by pulling off the entire volume of the dish and transferring it into a centrifuge tube. Keep the tube in the hood and allow the cells to settle to the bottom of the tube (about 5 minutes). Then, using a pipette, remove the supernatant from the tube and replace it with fresh, equilibrated KSR EB medium. Place the cells back onto the same dish.
18. Continue to change the medium every other day. The EBs will grow in size over time.
19. Alternatively, after 4 days, transfer the cells equally into 2 x 100 mm tissue culture treated dishes to allow for attachment. The attachment of EBs will allow for further differentiation of the cells into various cell types.
20. Keep the same schedule for fluid changes over time, however as the cells expand the volume per dish may need to be increased by 50% or 100%.
21. Allow the cells to expand for time points at 14 days and 21 days, or even longer. The entire EB dish can be harvested for analysis.

To generate EBs from mESCs, stem cells are passaged as usual but plated into non tissue culture-treated dishes, preventing attachment and allowing the cells to aggregate in suspension. These three-dimensional clusters initiate spontaneous differentiation, enabling researchers to evaluate the developmental potential and lineage competence of the stem cell population.

1. To prepare 100 mL of complete mESC EB Medium, aseptically mix the following components:
   
   

Component	Volume
KnockOut DMEM/F-12	83 mL
KnockOut Serum Replacement (KSR)	15 mL
MEM Non-Essential Amino Acids Solution, 10 mM	1 mL
GlutaMAX Supplement	1 mL

2. Complete mESC EB Medium can be stored at 2–8°C for up to 1 week.

1. To prepare 100 mL of complete mESC EB Medium with 10% FBS, aseptically mix the following components:

Component	Volume
mESC EB Medium	90 mL
FBS	10 mL

2. Complete mESC EB Medium with 10% FBS can be stored at 2–8°C for up to 1 week.

Attachment Factor (AF) dishes are required at Day 4 of the EB culture. Prepare one AF-coated plate for each sample of mESC EBs you plan to generate.

1. Cover the whole surface of a 100-mm tissue culture-treated dish with Attachment Factor (AF) solution and incubate the vessels for 30 minutes at 37°C or for 2 hours at room temperature.
2. Using sterile technique in a laminar flow culture hood, completely remove the AF solution from the culture vessel by aspiration just prior to use. Coated vessels may be used immediately or stored at room temperature for up to 24 hours.
   Note: It is not necessary to wash the culture surface before adding cells or medium.

1. Pre-warm the mESC EB Medium, Dulbecco’s PBS (DPBS) without calcium and magnesium, and Accutase solution to 37°C.
2. The EBs will be going into 100-mm tissue culture-treated dishes (i.e., adherent dishes) in order to separate mESC from MEF feeder cells. Prepare dishes by adding 10 mL of mESC EB Medium to each dish.
3. Aspirate the spent medium from the dish containing mESCs, and rinse the dish once with DPBS (e.g., 2 mL for one well of a 6-well plate).
4. Aspirate DPBS and add Accutase solution to the dish containing mESCs. Adjust the volume of Accutase solution for various dish sizes (Table 2).

5. Incubate the dish(es) for 1–2 minutes in a 37°C, 5% CO₂ incubator until individual single cells start to round up.
6. Add an appropriate amount of mESC EB Medium to each dish (Table 2) to stop dissociation reaction. Gently pipet the dissociated cells up and down sufficiently to disperse the colonies into a single-cell suspension. Make sure to pipet gently to minimize the formation of bubbles.
7. Transfer the mESC suspension from each well into a separate 15-mL conical tube and centrifuge the tube(s) at 250 × g for 5 minutes to pellet the cells.
8. Carefully aspirate the supernatant(s) from the mESC pellet(s).
9. Resuspend the pellet(s) with an appropriate amount (approximately 2 mL) of mESC EB Medium.
10. Add resuspended cells to each 100-mm prepared adherent dish containing 10 mL mESC EB Medium.
11. Incubate the adherent dish(es) for 40 minutes in a 37°C, 5% CO₂ incubator to separate the mESCs from the MEF feeder layer.
12. Carefully collect the mESCs that are detached from the MEF feeder layer and adjust the cell concentration to 5.5 × 10⁵ cells/mL with mESC EB Medium.
13. Plate 5 mL of the cell suspension (at 5.5 × 10⁵ cells/mL) in one 100-mm non-adherent dish (i.e., not tissue culture–treated).
14. Incubate the cells for 3 days in a 37°C, 5% CO₂ incubator to allow them to form EBs. Replace spent medium daily.

15. Plate EBs in mESC EB Medium supplemented with 10% FBS in an AF-coated dish.
16. Incubate EBs overnight in a 37°C, 5% CO₂ incubator to allow them to attach to the dish.
    Note: While cells are attaching, be careful when opening and closing the incubator doors to avoid disturbing the even distribution of cells on the surface of the wells.
17. Once the EBs are attached to the dish, aspirate medium and add mESC EB Medium (without FBS) to the dish. Return dish to the incubator and replace spent medium every other day.
18. At Day 14 after EB differentiation, stain the differentiated cells with antibodies against endodermal, mesodermal, and ectodermal markers.

Immunocytochemical staining is used to evaluate the ability of the mESCs to differentiate into the three primary germ layers. Stain the differentiated cells (at Day 14) with antibodies against the mesoderm marker smooth muscle actin, the ectoderm marker βIII-tubulin, and the endoderm marker α-fetoprotein (Table 3).

1. To prepare 10 mL of Blocking Buffer, aseptically mix the following components:

Component	Volume
DPBS without calcium and magnesium	7.2 mL
Goat serum	0.5 mL
1% Triton X-100	0.3 mL
50 mg/mL BSA	2.0 mL

2. Blocking Buffer can be stored at 2–8°C for up to 2 weeks.

1. To prepare 10 mL of DAPI Solution, aseptically mix the following components:

Component	Volume
DAPI	10 mg
DMSO	10 mL

2. Aliquot and store at –20°C. Dilute in DPBS without Calcium and Magnesium (1:1000) to make working solution.

1. Aspirate medium from the culture vessel.
2. Rinse the cells with DPBS (without calcium and magnesium) once.
3. Fix cells with 4% PFA for 15–20 min at room temperature.
4. Rinse cells with DPBS (without Calcium and Magnesium) three times for 5 minutes at room temperature.
5. Block cells in Blocking Buffer for 30 minutes at room temperature.
6. Add each primary antibody (βIII-tubulin, α-fetoprotein, and smooth muscle actin diluted in blocking buffer as indicated above in Table 3) separately to the cells. Use 6 mL (total volume) per 100-mm dish.
7. Incubate cells with primary antibodies at 4°C overnight.
8. Next day, rinse cells with DPBS (without calcium and magnesium) three times for 10 minutes at room temperature.
9. Treat the cells with the secondary antibody, Alexa Fluor 594 Goat Anti-Rabbit IgG, diluted in Blocking Buffer (1:1000). Use 6 mL per 100-mm dish.
10. Incubate cells with secondary antibody for 1 hour at room temperature.
11. Rinse cells with DPBS (without calcium and magnesium) twice for 10 minutes at room temperature.
12. Stain cells with DAPI working solution (diluted as instructed above) for 5 minutes.
13. Rinse cells with DPBS (without calcium and magnesium) for 10 minutes before visualizing.
14. ProLong Gold Antifade Reagent can be used at this time in the final wash step.

In the PSC Culture Medium, DMEM/F12 containing GlutaMAX Supplement can be substituted with the following alternatives:

• KnockOut DMEM/F-12

To prepare 100 mL of complete PSC Culture Medium using KnockOut DMEM/F-12, aseptically combine the components listed in the table below.

• KnockOut DMEM

To prepare 100 mL of complete PSC Culture Medium using KnockOut DMEM, aseptically combine the components listed in the table below.

In the mESC EB Medium, KnockOut DMEM can be substituted with the following alternatives:

• KnockOut DMEM/F-12

To prepare 100 mL of complete mESC EB Medium using KnockOut DMEM/F-12, aseptically combine the components listed in the table below.

1. Takahashi, K. et al. (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663–676. 
2. Takahashi, K. et al. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861–872. 
3. Yu, J. et al. (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science 318 :1917–1920. 
4. Maherali, N. et al. (2008) Guidelines and techniques for the generation of induced pluripotent stem cells. Cell Stem Cell 3:595–605. 
5. Li, W. et al. (2009) Generation of rat and human induced pluripotent stem cells by combining genetic reprogramming and chemical inhibitors. Cell Stem Cell 4:16–19. 
6. Liao, J. et al. (2009) Generation of induced pluripotent stem cell lines from adult rat cells. Cell Stem Cell 4:11–15. 
7. Dimos, J.T. et al. (2008) Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science 321:1218–1221. 
8. Aasen, T. et al. (2008) Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes. Nat Biotechnol 26:1276–1284. 
9. Park, I.H. et al. (2008) Generation of human-induced pluripotent stem cells. Nat Protoc 3:1180–1186.