Solutions
B Cell Medium
To prepare 100 ml of B cell medium mix the following:
RPMI with L-Glutamine 1X 88.9ml
Fetal bovine serum 10% 10ml
Penicillin-Streptomycin 100X 1ml
β-Mercaptoethanol 1000X 100μl
Interleukin 7 1000X 100μl
The medium can be stored at 4ºC for up to two weeks.
Cell Reprogramming Medium
To prepare 500 ml of N2B27 medium mix the following*:
DMEM/F12 231ml
Neurobasal medium 231ml
Penicillin-Streptomycin 100X 5ml
β-Mercaptoethanol 1000X 500μl
Sodium pyruvate 100X 5ml
NE-aminoacids 100X 5ml
Leukemia inhibitor factor 10000X 50μl
N2 supplement 100X 5ml
B27 supplement 50X 10ml
Interleukin 7 1000X 500μl
Interleukin 4 500X 1000μl
Interleukin 15 5000X 100μl
Doxycycline 500X**** 1000μl
- From day 2 onwards supplement the N2B27medium with 20% KSR, 3uM CHIR99021 and 1uM PD0325901.
****Prepare the reprogramming medium without inducer and supplement Doxycycline when needed.
S17/MEF Medium
To prepare 100 ml of OP9/S17 cell medium mix the following:
DMEM 89ml
Fetal bovine serum 20% 20ml
Penicillin-Streptomycin 100X 1ml
β-Mercaptoethanol 1000X 100μl
Interleukin 7, 4 and 15 Stock Solutions
To prepare 1ml of a 10μg/ml interleukin solution, mix the following:
Interleukin 10μg
Deionized water 1ml
Estradiol Stock Solution
To prepare 1mM b-Estradiol solution (10.000X), mix the following:
b-Estradiol 272ug
EtOH 1ml
Doxycycline Stock Solution
To prepare 1ml of a 50mg/ml Doxycycline solution, mix the following:
Doxycycline 50mg
Deionized water 1ml
Labeling Buffer
To prepare 250ml of labeling buffer, mix the following:
PBS 1X 240ml
FBS 10ml
EDTA 0.5M 1ml
B cell Isolation
One week before performing the reprogramming experiment purify ‘B cells’ from the bone marrow of two reprogrammable mice by sorting CD19 surface antigen positive cells, which consist of a mixture of pre-B and pro-B cells.
- Sacrifice two 8-16 week old reprogrammable mice/Oct4GFP mice by ventilating them with CO2.
- Aseptically remove femurs and tibiae.
- Use scissors to remove all tissues from the bones.
- Crush the bones in a mortar in 10 ml of labeling buffer.
- Thoroughly resuspend the cells by pipetting them up and down.
- Filter the cells with a 40μm cell strainer in a 50 ml conical tube.
- Centrifuge the cells for 5 minutes at 300xg, resuspend them in fresh labeling buffer and count them in a hemocytometer.
- Resuspend the cells in labeling buffer to a final concentration of 10X106 cells/ml.
- Add Fc-block to the cell suspension at a concentration of 0.1μg/106 cells and incubate for 10 minutes on ice.
- Add CD19-biotin conjugated antibody (0.2ug/106 cells) and incubate for 20 minutes on ice.
- Wash the cells with 10ml of labeling buffer, centrifuge for 5 minutes at 300xg. Resuspend them in 90μl of labeling buffer and 10μl of Streptavidin beads per 10 x106 cells and incubate for 20 minutes on ice.
- Wash the cells with 10ml of labeling buffer and proceed with purification using the MACS LS column.
- Place the LS column in the MACS separator.
- Wash the column with labeling buffer.
- Pipette 500μl of cells in the LS column allowing the suspension to run through and collect the effluent as negative fraction (this fraction is enriched for myeloid progenitors).
- Wash the column with 3ml of labeling buffer.
17 Repeat STEP 17 three times.
- Remove the column from the MACS separator and place it in a new 15ml collection tube.
Apply 5ml of labeling buffer and firmly flush out the cells using the plunger provided. This yields the CD19 positive B cell fraction.
Reprogramming of Bells
Retrovirus Production and B Cell Infection
- Two days before viral transduction seed 9x106 PlatE cells (Cell Biolabs) into two 10cm dishes. To detach PlatE cells wash the cells once with PBS and then add 1-3ml of TrypLE select cell dissociation reagent. After 3-5 minutes at 37°C add PlatE medium and count the cells in the hemocytometer.
- The following morning transfect PlatE cells with 20μg of C/EBP-ER-hCD4 plasmid using the calcium phosphate protocol.
- Replace the transfection medium after 8 hours with 6 ml of fresh B cell medium per 10cm plate. Culture the cells at 37°C, 5% CO2 incubator.
- After 18-20 hours of culture at 37°C collect the supernatant, filter it using a 0.45μm sterile filter, add 10ng/ml of IL-7 and use the filtered medium to infect the B cells. Use 2ml of supernatant to infect 2X106 B cells per well of a 6-weel plate. Add 6 ml of fresh B cell medium to the PlatE cells. Place the cells in a 37°C, 5% CO2 incubator.
24 hours later collect the supernatant of the PlatE cells and use it to infect the B cells a second time.
Plating of B cells on S17 Stromal Cells
We use S17 stromal cells for the first days of B cell expansion before the reprogramming is initiated.
- The day of the viral transduction experiment prepare gelatin-coated dishes. Then plate Mitomycin C inactivated S17 cells at a density of 3X104 cells/cm2 onto 10cm plates. To detach S17 cells, wash the cells with PBS and add the adequate amount of TrypLE select cell dissociation reagent to the cells. After 3-5 minutes, add S17 medium and count the cells in the hemocytometer.
- Collect the infected B cells from the 6 well plate and centrifuge them at 300xg for 5 min. Then aspirate the supernatant, and resuspend the cells in 10 ml of B cell medium. Plate the B cells on the inactivated S17 cells.
- Grow the cells 4 days or until they reach confluence (note that besides B cells that remain non-adherent, many of them move under the feeders, forming clusters of flattened cells with smooth edges that can easily be distinguished from the feeder cells).
- Collect the B cells, stain them with human CD4 antibody and sort hCD4+ transgene containing cells using a MACS column or a fluorescence activated cell sorter.
- The day before the sorting prepare gelatin-coated 12 well dishes. Plate Mitomycin C inactivated MEF cells at a density of 3X104 cells/cm2. To detach MEF cells add the adequate amount of TrypLE select cell dissociation reagent. After 3-5 minutes, add S17/MEF medium and count the cells in the hemocytometer.
- After the sorting of the hCD4+ B cells, count the cells using the desired method and seed the cells on MEF feeders at a density of 25.000 cells/cm2. Let the cells grow for two days.
- To activate C/EBPa add β-estradiol at a concentration of 100nM to the B cell medium.
- After 18h collect the supernatant and carefully wash the cultures twice with 500μl of PBS. Then centrifuge the supernatant/PBS at 300xg for 5 min.
- Resuspend the pellet in fresh reprogramming medium containing 2ug/ml of doxycycline and re-seed the cells into the original wells.
- Gently replace the medium every two days.
- Starting at day 1 Oct4GFP positive cells can be observed under a fluorescence microscope or recorded by FACS.
- After 2 days supplement the reprogramming medium with 20%KSR, 3uM CHIR99021 and 1uM PD0325901.
- Starting at day 4 Oct4GFP positive colonies with ES-like morphology appear.
- At day 8 change the medium to reprogramming medium without doxycycline to select transgene-independent iPS cell colonies.