Definitive Endoderm 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 critical. Use of suboptimal quality or very high passage pluripotent cells can result in decreased differentiation efficiency and/or increased cell death.

Supplies Required

Reagents Supplied in the StemXVivo™ Endoderm Kit (Catalog # SC019).

  • Endoderm Base Media Supplement
  • FGF basic
  • Activin A
  • Wnt-3a

Additional Reagents Required

  • RPMI Medium 1640
  • DMEM/F-12
  • GlutaMAX™
  • Penicillin-Streptomycin
  • Phosphate-Buffered Saline (PBS)
  • Cultrex® PathClear®Basement Membrane Extract Reduced Growth Factor (R&D Systems, Catalog # 3433-005-02 or equivalent)
  • MEF Conditioned Media (R&D Systems, Catalog # AR005 or equivalent)
  • Trypan Blue Stain
  • Accutase® (Innovative Cell Technologies, Catalog # AT104 or equivalent)
  • BSA, very low endotoxin
  • Sterile, deionized water

Materials

  • Human pluripotent stem cells (ATCC, Catalog # SCRC-2002 or equivalent)
  • 60 mm tissue culture dishes
  • 24-well culture plates
  • 15 mL centrifuge tubes
  • 50 mL centrifuge tubes
  • 0.2 μm syringe filter
  • 0.2 μm, 500 mL filter units
  • 10 mL syringes
  • Serological pipettes
  • Pipettes and pipette tips

Equipment

  • 37 °C, 5% CO2 humidified incubator
  • 37 °C Water bath
  • Centrifuge
  • Hemocytometer
  • Inverted microscope

Reagent and Material Preparation

0.1% BSA in PBS Dissolve 10 mg of BSA in 10 mL of PBS. Sterile filter the solution by syringe filter and store at 2 °C to 8 °C for up to 3 months.
Human FGF basic (1000X) Reconstitute the human FGF basic with 350 μL of sterile 0.1% BSA in PBS. Mix gently. Store at 2 °C to 8 °C for up to one month or aliquot and store at ≤-20 °C in a manual defrost freezer for up to 3 months. Avoid repeated freeze-thaw cycles.
Human Activin A (1000X) Reconstitute the human Activin A with 250 μL of sterile 0.1% BSA in PBS. Mix gently. Store at 2 °C to 8 °C for up to one month or aliquot and store at ≤-20 °C in a manual defrost freezer for up to 3 months. Avoid repeated freeze-thaw cycles.
Human Wnt-3a (1000X) Reconstitute the human Wnt-3a with 100 μL of sterile 0.1% BSA in PBS. Mix gently. Store at 2 °C to 8 °C for up to one month or aliquot and store at ≤-20 °C in a manual defrost freezer for up to 3 months. Avoid repeated freeze-thaw cycles.
Differentiation Base Media Add 25 mL of 10X Endoderm Base Media Supplement to 225 mL of RPMI, 2.5 mL of Penicillin/Streptomycin (optional), and 2.5 mL of GlutaMAX. Store at 2 °C to 8 °C for up to 2 weeks.
Differentiation Media I Dilute the FGF basic, Activin A, and Wnt-3a stock solutions 1000-fold in Differentiation Base Media. Prepare fresh as needed.
Differentiation Media II Dilute the FGF basic and Activin A stock solutions 1000-fold in Differentiation Base Media. Prepare fresh as needed.

Procedure

Stage I: Undifferentiated Cell Preparation

Coating Plates with Cultrex® Basement Membrane Extracts (BME)

  1. Thaw Cultrex BME on ice at 2 °C to 8 °C overnight.
  2. Dilute the Cultrex BME 40-fold in DMEM/F12. This can be stored at 2 – 8 °C for up to 2 weeks.
  3. Coat the desired number of plates with diluted Cultrex BME (approximately 2.5 mL per 60 mm plate and 0.5 mL per well of a 24-well plate) and incubate for 1 - 2 hours at room temperature.
  4. Remove the Cultrex BME solution immediately prior to plating the cells.
  5. Aliquot any remaining thawed Cultrex BME into pre-cooled tubes and store at ≤-20 °C.

Cell Dissociation

  1. Warm the MEF Conditioned Media to 37 °C.
  2. Remove the MEF Conditioned Media from the cells. Add 1 mL of Accutase cell detachment solution to each 60 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. Gently pipette over the plate until the cells have become detached.
  4. Gently pipette the cell suspension up and down to break up large cell clumps.
  5. Transfer the cell suspension to a 15 mL centrifuge tube containing 5 mL of conditioned media, and centrifuge for 4 minutes at 200 x g.

Cell Plating

  1. Resuspend the pellet in MEF Conditioned Media containing human FGF basic (1X) and count the viable cells using Trypan Blue and a hemocytometer.
  2. Plate the cells onto prepared BME-coated plates at a concentration of 1.17 x 106 cells/cm2. For example, plate 7 x 106 cells/60 mm plate. Because cells are being plated so densely, add extra media for growth overnight to ensure that the plates do not become too acidic. Use 7 - 8 mL of MEF Conditioned Media containing human FGF basic (1X) per 60 mm plate and 1.0 - 1.5 mL/well of a 24-well plate.
  3. Grow overnight at 37 °C and 5% C02. The next day, each plate should contain a tightly packed monolayer of cells.

  4. Note: As an alternative to plating cells at a high density and initiating differentiation the following day as described above, cells can be plated at a lower density and be grown to confluency prior to differentiation. This method may introduce some variability during differentiation.

Stage II: Day 1 of Differentiation

  1. Warm the MEF Conditioned Media to 37 °C.
  2. Remove the media from the plates 12 - 24 hours after initial plating and replace with fresh MEF Conditioned Media containing human FGF basic (1X). Use 5 - 6 mL of MEF Conditioned Media containing human FGF basic (1X) per 60 mm plate and 1.0 mL/well of a 24-well plate.
  3. Incubate cells at 37 °C and 5% C02 for a minimum of 2 - 4 hours prior to adding Differentiation Media I.
  4. Warm Differentiation Base Media to 37 °C.
  5. Prepare the required amount of Differentiation Media I. Due to the large quantity of cells in each plate, large volumes of media are required to ensure plates do not become too acidic. Use 7 mL of media per 60 mm plate and 1.5 mL per well of a 24-well plate.
  6. Remove the MEF Conditioned Media from each plate/well.
  7. Wash each plate/well once with sterile 1X PBS. Use approximately 3 - 4 mL per 60 mm plate and 0.5 - 1.0 mL/well of a 24-well plate.
  8. Add prepared Differentiation Media I to each plate and incubate overnight at 37 °C and 5% C02.

Stage III: Days 2 and 3 of Differentiation

  1. Approximately 12 - 16 hours after adding the Differentiation Media I, warm Differentiation Base Media to 37° C.
  2. Prepare the required amount of Differentiation Media II. Due to the large quantity of cells in each plate, large volumes of media are required to ensure plates do not become too acidic. Use 7 mL of media per 60 mm plate and 1.5 mL per well of a 24-well plate.
  3. Remove Differentiation Media I and replace it with Differentiation Media II.
  4. Repeat steps 2 - 4 within 8 - 12 hours (media should be replaced twice daily).
  5. Repeat steps 1 - 4 on Day 3.
  6. On Day 4, the cells are ready for further differentiation to downstream cell types or analysis by immunocytochemistry and/or flow cytometry.
Definitive Endoderm Differentiated BG01V Human Embryonic Stem Cells. BG01V human embryonic stem cells were differentiated using the StemXVivo Endoderm Kit (Catalog # SC019) and stained with A) a Goat Anti-Human SOX17 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1924), B) a Goat Anti-Human HNF-3 beta/FoxA2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2400), or C) a Goat Anti-Human SOX7 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF2766). In all images, cells were stained with the NorthernLights™ 557-conjugated Donkey Anti-Goat Secondary Antibody (Catalog # NL001; red) and counterstained with DAPI (blue). Endoderm differentiated cells were also stained with D) a PE-conjugated Mouse Anti-Human CXCR4 Monoclonal Antibody (Catalog # FAB173P; filled histogram) or a PE-conjugated Mouse IgG2B Isotype Control (Catalog # IC0041P; open histogram). High levels of SOX17 and low levels of SOX7 indicate endoderm differentiation.

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