Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

Ethics statement: Mouse cell lines are derived following the animal care guidelines of the Icahn School of Medicine at Mount Sinai, and should be done in compliance with any host institution.

1. Mouse Embryonic Fibroblast (MEF) Isolation of C57BL/6 Mice

1. Set up timed mating¹³. Once a vaginal plug is visualized, consider this Day 0.5.
2. Separate the plugged females on the plug date and check on them on Day 10-11 to confirm pregnancy.
3. On Day 13.5-14.5 euthanize pregnant female mice via CO₂ inhalation followed by cervical dislocation.
4. Soak pregnant female abdomen with 70% ethanol, dissect the abdominal cavity with sterile scissors and forceps down the midline, and surgically remove the uterine horns containing the embryos¹⁴. Take out the embryos gently while cutting off the remaining abdominal tissue.
   NOTE: Recover about 8 embryos with 4 on each side.
5. Place the uterine horns containing the embryos in a 10 cm dish with 5-10 ml of sterile chilled phosphate buffered saline (PBS).
6. With sterile scissors and forceps, make an incision along the uterine horns to isolate the sacs carrying the embryos. Transfer the sacs to a new 10 cm dish with 5-10 ml of sterile chilled PBS and place on ice.
7. Place a few of the sacs into a new 10 cm dish with 5-10 ml of sterile chilled PBS. Using sterile scissors and forceps gently cut open the sacs (without cutting too deep) in order to avoid piercing the embryos.
8. Separate the embryos from the placenta by cutting the umbilical cord.
9. Transfer the embryos to a new 10 cm dish containing 5-10 ml of chilled PBS and leave on ice while completing the remaining dissections.
10. Decapitate the embryos using sterile forceps. Carefully cut down the midline of the embryo using sterile scissors and forceps, starting from the decapitated area to open up the abdomen. Position forceps underneath the visceral organs (including the heart, liver, and lungs) and scrape them out¹⁵.
11. Cut the remaining tissue into small pieces using scissors and forceps and transfer to a 50 ml conical tube containing 10 ml of trypsin.
12. Pipet up and down with a 10 ml pipet 20-30 times to mix and dissociate the tissue.
13. Incubate the tissue and trypsin mix in a 37 °C water bath for 45 min, swirling occasionally.
14. Pipet up and down with a 10 ml pipet for about 5 minutes to mix the contents.
15. Add trypsinized cells to 3x volume of standard Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS), 10 µg/ml Penicillin/Streptomycin (P/S), and 1 mM L-Glutamine (L-GLUT) in 10 cm dishes (~10 ml) and culture at 37 °C until confluent (about 2-4 days). Culture about 1-2 embryos per 10 cm dish.
    NOTE: Freeze cells once plates are confluent. Frozen cells can be thawed and passaged 2 more times. Cells can alternatively be split 2 times prior to freezing, and then once more after being thawed.

2. Viral Production

1. Expand 293T cells in 10 cm plates with 10 ml DMEM with 10% FBS, 1% P/S, and 1% L-GLUT for transfection.
   1. Trypsinize 293T cells with 2 ml trypsin, incubate at 37 °C for 5 min, collect the cells into 15 ml tubes, spin at 300 x g, and plate appropriately (10 ml per 10 cm dish).
2. 24 hr prior to transfection, split confluent 10 cm plate of 293T cells 1:6 and seed in 10 cm gelatin coated (0.1% gelatin in PBS) plates.
   NOTE: 4 plates per virus will be required.
   1. To coat plates in gelatin, add at least 2 ml of the 0.1% gelatin solution to coat the bottom of 10 cm plates. Incubate at 37 °C for at least 20 min. Aspirate off gelatin prior to adding cells to the plates.
3. In a 15 ml tube, add 84 µg plasmid (e.g. Gata2, Gfi1b, cFos, or Etv6 (sub-cloned into the pFUW-TetO vector¹⁶) or FUW-M2rtTA¹⁷), 84 µg psPAX2, and 42 µg pMD2.G. Add water (H₂O) to make up the volume to 2 ml. Prepare a tube for each factor (e.g. Gata2, Gfi1b, cFos, Etv6 and FUW-M2rtTA).
4. Add 250 µl of 2M CaCl₂ to each tube containing the 2 ml mixture composed of 84 µg of the chosen gene (Gata2, Gfi1b, cFos, Etv6 or FUW-M2rtTA) + 84 µg psPAX2 + 42 µg pMD2.G + H₂O.
5. Using a Pasteur pipet inserted into the pipet-aid, release bubbles into the 2.25 ml DNA + H₂O + 2 M CaCl₂ mixture. As the bubbles are being produced, place the tip of a P1000 against the glass pipet and slowly eject 2 ml of 100 mM N,N-bis (2-hydroxyethyl)-2-aminoethane sulfonic acid (BES) saline down the Pasteur pipet 1 ml at a time.
6. Incubate all tubes in the culture hood at room temperature for at least 15 min.
   NOTE: The 4.25 ml mixture (now DNA + H₂O + 2M CaCl₂ + BES saline) will look slightly cloudy.
7. As the mixture incubates, aspirate media off 293T plates and add 10 ml DMEM + 10% FBS + 1 mM L-Glutamine + 25 nM chloroquine.
   NOTE: This media does not contain P/S.
8. After at least 15 min of incubation (or until changing media on 293T cells), add the 4.25 ml DNA + H₂O + 2 M CaCl₂ + BES saline mixture drop wise to the 293T cells, distributing evenly across 4 cell plates per tube (i.e. slightly more than 1 ml of the mixture per plate).
9. Incubate plates overnight (O/N) at 37 °C.
10. 24 hr post incubation, replace media on all plates with 4 ml standard DMEM (see step 1.15).
11. Keep cells in a 32 °C incubator from now on.
12. 24 hr after step 2.10, collect media into separate 50 ml tubes. Replace the collected media from each plate with 4 ml of standard DMEM.
    ​NOTE: 4 plates per initial tube mixture results in 16 ml of collected media.
    1. After 12 hr of incubation, collect media again into the same 50 ml tubes and replace with another 4 ml of standard DMEM.
    2. After 12 hr of incubation, collect the media and add to the same 50 ml tubes.
       NOTE: each tube should now contain about 48 ml of media containing virus.
13. To concentrate the collected virus, first filter the collected media through 0.45 µM low-protein binding filters.
14. Pour the filtered media containing virus into viral concentration centrifuge tubes (about 16 ml at a time).
15. Spin at 4,000 x g at 4 °C for 25 min and remove flow-through.
    NOTE: A small volume of concentrated media and virus will be visible in the filter.
16. Add another 16 ml of filtered virus-containing media to the concentrated media + virus volume remaining in the filter and mix by inverting the tube.
17. Spin tube again at 4,000 x g at 4 °C for 25 min and discard flow-through.
18. Add remaining filtered collection to the concentrated media + virus volume left in the filter and mix by inverting the tube.
19. Spin once more at 4,000 x g at 4 °C for 10-20 min (depending on the volume in the filter) and ensure that the remaining volume of concentrated media + virus left in the filter is about 1 ml. If greater than 1 ml of concentrated media + virus remains in the filter, spin again at 4,000 x g for 1 min and repeat until 1 ml is left in the filter.
20. Aliquot 200 µl of the concentrated virus into 1.5 ml tubes and freeze at -80 °C or use immediately.

3. MEF Reprogramming

1. Pre-coat 6 well-plates with 0.5 ml of 0.1% gelatin in PBS per well, ensuring that the gelatin covers the entire area of the well. Place in a 37 °C incubator and incubate for at least 20 min. After incubation, remove the plates and aspirate the gelatin.
2. Plate MEFs with standard DMEM at a density of 25,000 cells per well (150,000 cells per 6-well plate) on the gelatinized 6-well plate(s) from step 3.1 and allow to settle O/N in a 37 °C incubator.
3. Prepare a 100 µl virus cocktail by mixing 50 µl M2rtTA and the remaining 50 µl a mix of Gata2, Gfi1b, cFos, and Etv6 (12.5 µl each).
4. Aspirate media from MEFs and add 2 mL standard DMEM with 8 µg/ml hexadimethrine bromide.
5. Transduce each well of the MEF plates with 15 µl of the virus cocktail and incubate O/N at 37 °C.
   NOTE: This is Day 0 of the reprogramming process.
6. On Day 1, after 16-20 hr of incubation, replace media with 2 ml fresh standard DMEM supplemented with 1 µg/ml DOX per well.
7. Prepare hematopoietic culture media supplemented with hydrocortisone (10^-6 M), 100 ng/ml stem cell factor (SCF), 100 ng/ml Fms-related tyrosine kinase 3 ligand (Flt3L), 20 ng/ml interleukin-3 (IL-3), 20 ng/ml interleukin-6 (IL-6), and 1 µg/ml DOX.
8. On Day 4 aspirate media from each well and wash cells with 1 ml of PBS per well.
9. Aspirate PBS, dissociate cells using 1 ml of 0.05% trypsin per well and collect the cells.
10. Count cells with a hemocytometer, spin at 300 x g for 5 min, resuspend in 12 ml of hematopoietic media described in step 3.7 and plate 60,000 cells per 6-well plate on 0.1% gelatin coated plates (2 ml per well, 10,000 cells per well).
11. Replace media with fresh supplemented hematopoietic culture media every 6 days for the duration of culture (35 to 36 days).
12. Analyze either intermediate cells or emerging hematopoietic-like cells via immuno-staining¹², FACS analysis^8,12, qRT-PCR¹², and mRNA sequencing¹² to confirm the acquisition of hemogenic endothelial or HSC-like markers and gene expression.

4. Placental Aggregation and Colony Forming Unit (CFU) Assays in Methylcellulose

1. Dissect placentas from pregnant C57BL/6 mice as previously described¹⁸ at E12.5 and keep tissue on ice.
2. To start placenta dissociation¹⁹, wash placental tissue in 2-5 ml of 0.2% collagenase type I in PBS with 20% FBS and mechanically dissociate with an 18G needle fitted into a 5 ml syringe by passaging the placenta and PBS through the needle at least 3 times.
3. After mechanical dissociation, keep placental cells in 2-5 ml of collagenase solution at 37 °C for 1.5 hr.
4. Passage cells through 20-25 G needles fitted onto 5 ml syringes several times to further mechanically dissociate the placental cells.
5. Filter single cell suspensions through 70 µm cell strainers.
6. Count cells with a hemocytometer and irradiate at 2,000 cGy for mitotic inactivation²⁰.
7. Add 10 ml of hematopoietic culture media supplemented with 100 ng/ml SCF, 100 ng/ml Flt3L, 20 ng/ml IL-3, 20 ng/ml IL-6, and 10 ng/ml thrombopoietin (TPO) to a 10 cm dish.
   NOTE: DOX is not required at this stage.
8. Place a 0.65 µm filter directly on hematopoietic culture media in the dish, allowing it to float to create a liquid-gas interface and set the dish aside.
9. Dissociate day 25 transduced MEFs (derived from step 3.11) as described in steps 3.8-3.10 and mix 33,000 of the transduced MEFs with 167,000 cells of the placental aggregates from step 4.6 (1:6 ratio).
10. To generate an aggregate^18,21, first spin down the MEF and placental cell mix from step 4.9 for 5 min at 300 x g. Aspirate the media and resuspend the pelleted cells in 30-50 µl of standard DMEM using a P200 pipette, drawing the single cell suspension into the 200 µl nonbevelled pipette tip.
11. Occlude the pipette tip with paraffin film. Place the blocked tip into a 15 ml centrifuge tube and spin down for 5 min at 300 x g so a pellet forms at the covered end of the tip.
12. Remove the tip from the 15 ml tube carefully and place the tip onto the end of the P200 pipette. Discard the paraffin film and extrude the cell pellet onto the floating filter from step 4.8.
    NOTE: Plate at most 3 aggregates per filter.
13. Culture the aggregates on the floating 0.65 µm filters for 4-5 days in 37 °C with 5% CO₂.
14. Collect the aggregates (at most 3 per filter) with a cell scraper, combine them, and digest with 0.2% collagenase, following the same protocol as done with the whole placentas to dissociate the cells (steps 4.2-4.5).
15. After dissociation, wash the aggregates with 2-5 ml of PBS supplemented with 5% FBS and spin down cells at 300 x g for 5 min.
16. Resuspend the aggregates in 500 µl of DMEM without FBS, P/S, or L-GLUT. Take this 500 µl resuspension and add it to 3 ml 1% methylcellulose media supplemented with 10 ng/ml of TPO, carefully avoiding the formation of bubbles. Plate equal volumes of this mixture in 3 35 x 10 mm non-treated culture dishes for CFU assays¹².
17. As a control, culture irradiated placental aggregates without mixing in reprogrammed MEFs 4-5 days and place in CFU assays as described above in steps 4.7-4.16.
    NOTE: These aggregates alone do not form colonies.
18. Count hematopoietic colonies manually under the microscope after 10-14 days of culture at 37 °C with 5% CO₂.
19. Cytospin²² picked colonies to show morphological phenotype of single cells.