Endoderm, Ectoderm, and Mesoderm Differentiation of BG01V Human Embryonic Stem Cells

This protocol is designed for BG01V human embryonic stem (hES) cells grown in Mouse Embryonic Fibroblast (MEF) Conditioned Media (Catalog # AR005). If using different cell lines or growth media, the protocol below may need to be modified.

The quality of the human pluripotent cells used in the differentiation is imperative. Use of suboptimal quality or very high passage pluripotent cells can result in decreased differentiation efficiency and/or increased cell death.

Reagents supplied in the Human Pluripotent Stem Cell Functional Identification Kit (Catalog # SC027B)

• Differentiation Base Media Supplement (50X)
• Endoderm Differentiation Supplement I
• Endoderm Differentiation Supplement II
• Ectoderm Differentiation Supplement
• Mesoderm Differentiation Supplement
• Goat anti-human SOX17
• Goat anti-human Otx2
• Goat anti-human Brachyury

Other supplies required

Materials

• Human pluripotent stem cells
• 24-well culture plates
• 12 mm cover slips
• 15 mL centrifuge tubes
• 50 mL centrifuge tubes
• 0.2 μm syringe filter
• 10 mL syringe
• Pipettes and pipette tips
• Serological pipettes
• Glass slides
• Fine pointed curved forceps

Reagents

• RPMI
• BSA, very low endotoxin
• D-MEM/F-12 (1X)
• GlutaMAX™
• Penicillin-Streptomycin (optional)
• Phosphate Buffered Saline (PBS)
• Cultrex^® PathClear^® BME Reduced Growth Factor Basement Membrane Extract (Catalog # 3433-010-01 or equivalent)
• Recombinant human FGF basic (Tissue culture grade; Catalog # 4114-TC or equivalent)
• MEF Conditioned Media (Catalog # AR005 or equivalent)
• Trypan Blue Solution
• Accutase^® (Innovative Cell Technologies, Catalog # AT104 or equivalent)
• 95% Ethanol
• 4% Paraformaldehyde in PBS
• 1% BSA in PBS
• 0.3% Triton^® X-100, 1% BSA, 10% normal donkey serum in PBS
• 1% BSA, 10% normal donkey serum in PBS
• Mounting medium
• Secondary developing reagents (Catalog # NL001 or equivalent)
• Deionized or distilled water

Equipment

• 37 °C and 5% CO₂ incubator
• Centrifuge
• Hemocytometer
• Inverted microscope
• 37 °C water bath
• Fluorescence microscope

REAGENT AND MATERIAL PREPARATION

Procedure

UNDIFFERENTIATED CELL PREPARATION

Coating Plates with Cultrex BME

1. Thaw Cultrex PathClear BME (Catalog # 3433-010-01) on ice at 2 °C to 8 °C overnight.
2. Aliquot thawed Cultrex PathClear BME into pre-cooled tubes and store at ≤ -20 °C.
3. Thaw the aliquot on ice at 2 °C to 8 °C overnight.
4. Dilute Cultrex PathClear BME 1:40 in D-MEM/F-12. This can be stored at 2 °C to 8 °C for up to 2 weeks.
5. Place a sterile coverslip (sterilized with 95% ethanol and flamed) in each well of a 24-well plate.
6. Coat the desired number of wells with diluted Cultrex PathClear BME (0.5 mL/well of a 24-well plate) and incubate for 1 - 2 hours at room temperature.
7. Remove the Cultrex PathClear BME solution immediately prior to plating the cells.

Cell Dissociation

1. Warm the MEF Conditioned Media to 37 °C.
2. Remove the existing media from the cells. Add 1 mL of Accutase solution to each 60 mm plate or 3 mL to each 100 mm plate. Incubate at room temperature for 2 - 5 minutes or until cells begin to slough off the plate. If using cells from several plates, work in small batches (1 - 2 plates at a time) so cells are not exposed to the Accutase for an extended period of time.
3. Pipette gently over the plate until the cells become detached.
4. Gently pipette the cell suspension up and down to break up large cell clumps.
5. Remove the cell suspension to a 15 mL centrifuge tube containing 5 mL of MEF Conditioned Media (or 12 mL if using a 100 mm plate) and spin at 200 x g for 4 minutes.

Cell Plating

1. Resuspend the pellet in MEF Conditioned Media containing 4 ng/mL of FGF basic, and count the viable cells using Trypan Blue and a hemocytometer.
2. Plate cells onto prepared Cultrex PathClear BME-coated plates at a concentration of 1.1 x 10⁵ cells/cm². For example, plate 4.5 x 10⁶ cells divided among all wells of a 24-well plate. If your cells routinely grow slowly, the initial plating density can be increased.
3. Grow overnight at 37 °C and 5% CO₂. The next day each plate should be approximately 50% confluent. If cells are not 50% confluent, replace media with fresh media and culture until they reach 50% confluency.
4. Proceed to differentiation.

ENDODERM DIFFERENTIATION

Endoderm Differentiation (Day 1)

1. Warm Differentiation Base Media to 37 °C.
2. Prepare required amount of Endoderm Differentiation Media I. Use 1 mL of media/well of a 24-well plate.
3. Remove MEF Conditioned Media from each plate/well.
4. Add prepared Endoderm Differentiation Media I to each plate and incubate overnight at 37 °C and 5% CO₂.

Endoderm Differentiation (Days 2 and 3)

1. Approximately 16 - 24 hours after adding Endoderm Differentiation Media I, warm the Differentiation Base Media to 37 °C.
2. Prepare required amount of Endoderm Differentiation Media II. Use 1 mL per well of a 24-well plate.
3. Remove Endoderm Differentiation Media I and replace it with prepared Endoderm Differentiation Media II.
4. Repeat steps 1 - 3 on Day 3.
5. On Day 4, cells are ready for analysis by immunocytochemistry. Proceed to the Fixing and Staining Procedure.

ECTODERM DIFFERENTIATION

1. Warm Differentiation Base Media to 37 °C.
2. Prepare required amount of Ectoderm Differentiation Media. Use 1 mL of media/well of a 24-well plate.
3. Remove MEF Conditioned Media from each plate/well.
4. Add prepared Ectoderm Differentiation Media to each plate and incubate overnight at 37 °C and 5% CO₂.
5. Repeat steps 1 - 4 on Days 2 and 3.
6. On Day 4, cells are ready for analysis by immunocytochemistry. Proceed to the Fixing and Staining Procedure.

MESODERM DIFFERENTIATION

1. Warm Differentiation Base Media to 37 °C.
2. Prepare required amount of Mesoderm Differentiation Media. Use 1 mL of media/well of a 24-well plate.
3. Remove MEF Conditioned Media from each plate/well.
4. Add prepared Mesoderm Differentiation Media to each plate and incubate overnight at 37 °C and 5% CO₂.
5. Repeat steps 1 - 4 after 12 - 16 hours.
6. Approximately 24 - 36 hours after the initial differentiation, cells are ready for analysis by immunocytochemistry. Proceed to the Fixing and Staining Procedure.

FIXING AND STAINING PROCEDURE

1. Wash cells twice with PBS (1 mL/well of a 24-well plate).
2. Fix the cells with 4% paraformaldehyde in PBS for 20 minutes at room temperature.
3. Wash the cells 3 times with 1% BSA in PBS for 5 minutes (0.5 mL/well of a 24-well plate).
4. Permeabilize and block cells with 0.3% Triton X-100, 1% BSA, and 10% normal donkey serum in PBS at room temperature for 45 minutes (0.5 mL/well of a 24-well plate).
5. During the blocking, dilute the appropriate reconstituted primary antibody in PBS containing 0.3% Triton X-100, 1% BSA, and 10% normal donkey serum to a final concentration of 10 μg/mL.
   • Goat anti-human SOX17 (Endoderm)
   • Goat anti-human Otx2 (Ectoderm)
   • Goat anti-human Brachyury (Mesoderm)


6. Note: A negative control should be performed using PBS containing 0.3% Triton X-100, 1% BSA, and 10% normal donkey serum with no primary antibody.


7. After blocking, incubate cells with diluted primary antibody (300 μL/well of a 24-well plate) for 3 hours at room temperature or overnight at 2 °C to 8 °C.
8. Wash the cells 3 times with 1% BSA in PBS for 5 minutes (0.5 mL/well of a 24-well plate).
9. Dilute the secondary antibody [e.g. NorthernLightsTM 557-conjugated Donkey Anti-Goat Secondary Antibody (Catalog # NL001) at 1:200 in PBS containing 1% BSA.
10. Incubate cells with diluted secondary antibody in the dark for 60 minutes at room temperature (300 μL/well of a 24-well plate).
11. Wash the cells 3 times with 1% BSA in PBS for 5 minutes (0.5 mL/well of a 24-well plate).
12. Cover the cells with PBS (1 mL/well of a 24-well plate) and visualize with a fluorescence microscope.
13. Alternatively, aspirate PBS and add distilled or deionized water (0.5 mL/well of a 24-well plate). Carefully remove coverslips with forceps and mount cell-side down onto a drop of mounting medium on a glass slide.
14. Slides are ready for microscopic observation.

Endoderm, Ectoderm, and Mesoderm Differentiation of Human Pluripotent Stem Cells. BG01V human embryonic stem cells were differentiated to endoderm, ectoderm, and mesoderm using the media supplements included in the Human Pluripotent Stem Cell Functional Identification Kit (Catalog # SC027B). The kit also contains SOX17 (endoderm), Otx2 (ectoderm), and Brachyury (mesoderm) antibodies for the confirmation of differentiation status.

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