Induction of rEpiLCs and rPGCLCs

Induction of primordial germ cell-like cells from pluripotent stem cells in rats

Materials

DMEM (Gibco, cat.no. 11995-065)

Fetal bovine serum, FBS (Sigma-Aldrich, cat.no. 173012)

GlutaMAX (Thermo Fisher, cat.no.35050-061)

PBS (Wako, cat.no. 164-23551)

DMEM/F12, HEPES (Thermo Fisher Scientific, cat. no. 11330-032)

Neurobasal Medium (Thermo Fisher Scientific, cat. no. 21103-049)

N-2 supplement (Thermo Fisher Scientific, cat. no.17502-048)

B-27 supplement (Thermo Fisher Scientific, cat.no. 17504-044)

CHIR99021 (Axon Medchem, cat.co. Axon1386)

PD0325901 (Axon Medchem, cat.co. Axon1408)

ESGRO Recombinant Rat LIF Protein (rLIF), (Sigma-Aldrich, cat.no. ESG2207)

Dimethyl sulfoxide (DMSO), (Sigma-Aldrich, cat. no. D2650)

Trypisin-EDTA (0.25%), phenol red (Gibco, 25200-056)

KnockOut Serum Replacement (KSR), (Gibco, cat.no. 10828-010)

FGF-Basic (bFGF), Human, Recombinant (bFGF), (PEPRO TECH, cat. no. 100-18B)

Activin A, Human/Murine/Rat, Recombinant (PEPRO TECH, cat. no. 120-14)

SCF recombinant mouse, E.coli-derived (R&D systems, cat.no. 455-MC)

EGF, Human recombinant (R&D systems, cat. no. 236-EG)

BMP-4 (E.coli derived), Human, Recombinant (PEPRO TECH, cat. no. 120-05ET)

Penicillin-Streptomycin Solution (P/S) x100, (Wako, cat. no. 168-23191)

Tris-HCl (pH7.6) (Wako, cat. no. 208-14691)

Bovine Albumin Fraction V, BSA (7.5% solution), (Gibco, cat. no. 15260-037)

CultureSure Sterile Water (Wako, cat. no. 039-24155)

Gelatin solution, Type B, 2% in H₂O (Sigma-Aldrich, cat. no. D2650)

Cells

Mouse Embryonic Fibroblast (MEF) 

Inactivation treatment, such as Mitomycin-C or gamma irradiation, should be performed before use.

Rat embryonic stem cells (rESCs) 

Passaging cells: Start from 2 – 4 × 10⁴ cells per well of 12 well plate.

Frozen stock: Start from more than 2 – 3 × 10⁵ cells per well of 12 well plate.

Equipment

Nunc Cell-Culture Treated Multidishes, 12-well (Thermo Fisher Scientific, cat.no. 150628)

Nunclon Sphera 96-well, Nunclon Sphera Treated, U-shaped-Bottom Microplate (Thermo Fisher Scientific, cat. no.174929)

Nunc Cell-Culture Treated Multidishes, 4 well (Thermo Fisher Scientific, cat. no.176740)

Cell culture plate, 24 well, Flat bottom (TTP, cat. no. 92424)

LUNA-II Automated Cell Counter (Logos Biosystems, cat.no. L40001)

LUNA Cell Counting Slides (Logos Biosystems, cat.no. L12003)

BioLite 15 ml centrifuge tube (Thermo Fisher Scientific, cat. no. 339656)

Reagents

0.1% Gelatin/PBS solution Mix 15 ml of Gelatin solution and 285 ml of PBS. Sterilize the solution by filtration. This solution can be stored at room temperature.

MEF medium Mix 100 ml of DMEM with 11 ml of FBS, 1.1 ml of GlutaMAX and 1.1 ml of P/S. Store the medium at 4℃ up to 3 weeks.

N2B27 medium Mix 50 ml of D-MEM/F12 with 50 ml of Neurobasal Medium, 1 ml of B-27 serum free supplement, 500 µl of N-2 serum free supplement, 500 µl of GlutaMAX and 500 µl of P/S. Store the medium at 4℃ up to 3 weeks.

CHIR99021 stock solution Add358.2 µl of DMSO to 5 mg of CHIR99021. If it is hard to dissolve at room temperature, warm the bottle in the water bath at 37℃. The concentration becomes 30 mM. Make 4 µl aliquots and store them at -20℃.

PD0325901 stock solution Add 415 µl of DMSO to 2 mg of PD0325901. The concentration becomes 10 mM. Make 4 µl aliquots and store them at -20℃.

bFGF stock solution Add 500 µl of Tris-HCl (pH7.6) to 50 µg of bFGF. Subsequently plus 500 µl of 0.1%BSA/Tris-HCl (pH7.6). The concentration becomes 50 µg/ml. Make 10 µl aliquots and store them at -20℃.

Activin A stock solution Add 100 µl of DW and subsequently plus 100 µl of 0.1% BSA/DW. The concentration becomes 500 µg/ml. Make 10 µl aliquots and store them at -20℃.

SCF stock solution Add 500 µl of 0.1%BSA/PBS to 50 µg of SCF. The concentration becomes 100 µg/ml. Make 10 µl aliquots and store them at -20℃.

EGF stock solution Add 1 ml of 0.1% BSA/DW into 200 µg of EGF. The concentration becomes 200 µg/ml. Make 10 µl aliquots and store them at -20℃.

BMP4 stock solution Add 500 µl of 5 mM HCl to 100 µg of BMP4. Subsequently plus 0.1% BSA/5 mM HCl. The concentration becomes 100 µg/ml. Make 25 µl aliquots and store them at -20℃.

2i + rLIF medium Mix 40 ml of N2B27 medium with 4 µl of CHIR99021 stock solution, 4 µl of PD0325901 stock solution and 4 µl of rLIF. Store the medium at 4℃ up to 2 weeks.

Rat epiblast-like cell (rEpiLC) medium Mix 10 ml of N2B27 medium with 10 µl of KSR, 2.4 µl of bFGF stock solution, 0.4 µl of Activin A stock solution. This medium can be stored at 4℃ for a few days. Use up the medium in each experiment.

Rat primordial germ cell-like cell (rPGCLC) medium Mix 9.5 ml of N2B27 medium with 500 µl of KSR, 1 µl of rLIF, 10 µl of SCF stock solution, 2.5 µl of EGF stock solution, 50 µl of BMP4 stock solution. This medium can be stored at 4℃ for a few days. Use up the medium in each experiment.

Procedure

A. Culture of rESCs.

1. One day before, culture the MEF cells to the 12-well plate with 0.1% Gelatin/PBS solution. After 5 - 10 min incubation at room temperature, aspirate the solution.

2. Seed the MEF at 5 × 10⁵ cells per well to the gelatin coated plates. Culture the plate at 37℃ in a 5%CO₂ incubator. 

3. Next day, aspirate the MEF medium. Rinse with 1 ml of PBS and aspirate. Pipette 1 ml of 2i + rLIF medium into the well and incubate the plate untill rESCs seeding.

4. Seed the frozen-thawed rESCs at 2 - 4 × 10⁴ cells per well.

5. After 2 days, add 1 ml of 2i + rLIF medium per well. Culture 2 - 3 days before induction.

B. Induction of rEpiLCs.

1. Set the medium to the 96-well U bottom plate. Pipette 100 µl of rEpiLC medium per well while avoiding corner and edge. Pipette 100 µl of PBS into corner, edge and side wells to avoid evaporation of rEpiLC medium. Incubate the plate at 37℃ in a 5%CO₂ incubator until before use. 

2. Prepare the rESCs for rEpiLC induction. Discard upper 1 ml of 2i + rLIF medium from the well of rESC culture the plate.

3. Shake the plate gently side to side then collect rESCs with left medium into 15 ml tube. Rinse the well with 1 -2 ml of PBS and collect then into the same 15 ml tube.

4. Centrifuge the tube at 280 × g, 20℃, for 5 min.

5. Aspirate the medium while avoiding cell pellets.

6. Pipette 200 µl of 0.25% Trypsin/EDTA and incubate at 37℃ for 3 min in the water bath.

7. Add 2 ml of MEF medium and pipette gently to stop the reaction.

8. Centrifuge the tube at 280 × g, 20℃, for 5 min.

9. Aspirate the medium while avoiding cell pellets.

10. Pipette 200 µl of N2B27 medium and suspend the cell pellets.

11. Count the cell number. Seed 4 × 10³ cells per well into the rEpiLC containing well on the 96-well U bottom plate. Culture the plate at 37℃ in a 5%CO₂ incubator. Normally, culture the cells 48 – 72h for rEpiLC induction.

12. Next day, add 100 µl of rEpiLC medium per well. 

C. Induction of rPGCLCs.

1. Set the medium to the 96-well U bottom plate. Pipette 100 µl of rPGCLC medium per well while avoiding corner and edge. Pipette 100 µl of PBS into corner, edge and side wells. Incubate the plate at 37℃ in a 5%CO₂ incubator until before use. 

2. Prepare 200 µl pipette tip or glass capillaries. Cut the tip horizontally and round out it with gas burner. Rinse the equipment with N2B27 medium to prevent attaching the cells. 

3. Pipette 500 µl of N2B27 medium into the two wells of 4 well of 24 well plates. Collect rEpiLCs into the N2B27 medium under the Stereomicroscope.

4. Rinse the rEpiLCs and transfer to the second wells containing N2B27 medium.

5. Seed the rEpiLCs into rPGCLC medium containing wells on the 96-well U bottom plate. Use one rEpiLC spheroid per well. Culture the plate at 37℃ in a 5%CO₂ incubator.

6. After 2 days, add 100 µl of rPGCLC medium per well.

7. Confirm rPGCLC specification after 3 - 4 days after induction by immunofluorescence staining or flow cytometer.