Human Fibroblast to iMN Reprogramming

This protocols outlines the reprogramming of human dermal fibroblasts (HDFs) to induced motor neurons (iMNs).

This protocol outlines the timeline of Human Fibroblast to iMN reprogramming and links to protocols as they are appropriate in the timeline.

Days 1 - 6: Production and transduction of viruses encoding reprogramming factors

See here for the complete 293T virus production protocol.

At minimum, iMN reprogramming requires the transduction of motor neuron-specific transcription factors. Typically, the oncogenes (p53DD, hRasG12V, c-MYC, myrAKT, BCL2) are also introduced to enhance reprogramming efficiency. The following plasmids are frequently used as sources of motor neuron TFs or oncogenes and are encoded on retroviral backbones compatible with production 273Ts.

pKG number

Plasmid Name

pKG00121

pMXs-Ascl1

pKG00010

pMXs-Brn2

pKG00003

pMXs-Myt1L

pKG00006

pMXs-Ngn2

pKG00009

pMXs-Isl1

pKG00008

pMXs-Lhx2

pKG00002

pMXs-NeuroD1

pKG01169

pMXs-NIL

pKG01574

pMXs-LNI3xHA

pKG00060

pMXs-p53DD

pKG00121

pMXs-hRasG12V

pKG00747

pMXs-myrAkt-P2A-BCL2

pKG00751

pMXs-cMyc

pKG03523

pMXs-cMyc-IRES-p53DD

pKG03525

pMXs-cMyc-IRES-SNAP-p53DD

Day 1:

  1. Seed 6 million 293Ts in each gelatin-coated 10 cm plate for each virus to be made.

    • Each 10 cm plate makes 200 uL of concentrated virus. If more virus is needed, additional 10 cm plates should be seeded.

Tip

If starting from frozen, start growing 293Ts 1 week prior - they will be slow growing at first (don’t change culture medium during the first 3 days). Split 293Ts 4X-6X every 2-3 days when culture reaches 70-90% confluency.

You will likely need 2 to 3 T-182 flasks of 293Ts per human reprogramming experiment.

  1. Thaw HDFs into a gelatin coated T-182 so they can recover over several days. When culturing HDFs, it is recommended to use DMEM + 15% FBS + non-essential amino acids.

Day 2:

  1. Transfect transfer plasmids (containing your gene of interest), packaging, and envelope plasmids. Do this early in the day, as the media is changed 6-8 hours after transfection. See here for the complete 293T virus protocol.

  2. Media change 293Ts 6-8 hours after transfection. Remove media and add back 6.5 mL of DMEM + 10% FBS + 25 mM HEPES.

Day 3:

Warning

Be sure to use BSL2+ precautions when working with the human-infectible retrovirus containing oncogenes.

  1. Collect virus-containing media 22-24 hours after the previous media change in 50 mL conicals. Two plates can be combined into 1 tube. Replace with fresh media so that you can collect more virus the next day. Store collected virus in the 4℃ refrigerator overnight.

Day 4:

  1. Repeat the virus collection ~24 hours later, collecting virus in the same tubes.

  2. Filter with a 0.45 um filter into a new 50 mL conical.

  3. Add 1/3 volume of Lenti-X concentrator (e.g., for 36 mL virus, add 12 mL Lenti-X). Mix by inverting several times.

    Tip

    There is enough room in one 50 mL conical to collect 3 plates of virus for 2 days and add the appropriate volume of Lenti-X.

    BAD prefers to add Lenti-X to the new 50 mL conicals and then filter the virus directly into the Lenti-X.

  4. Store virus at 4°C overnight to precipitate the virus.

  5. Seed HDFs at 3k/96-well (for 35dpi reprogramming) or 8k/48-well (for a 7dpi CTV experiment) onto gelatin coated plates.

Day 5:

  1. Centrifuge precipitated virus at 1500 x g at 4°C for 45 minutes (use the lower centrifuge) to pellet the virus.

  2. Remove supernatent and resuspend in 200uL per 10 cm plate with DMEM + 15% FBS + non-essential amino acids (HDF media).

  3. Dilute virus according to calculations in fresh HDF media and polybrene. Use 1 uL of each concentrated virus per 96-well or 2.6 uL per 48-well.

  4. To improve infection efficiency, spin the plate at 1500 x g for 90 minutes at 32°C as described. Be sure to cover the centrifuge buckets with the plate spinner tray caps.

Day 6 (1 day post infection):

  1. ~24 hours after transduction, remove virus-containing media and replace with fresh HDF media.

  2. If you are interested in early proliferation during reprogramming, perform CellTrace Staining on ths day.

Days 7-11 (2-6 days post infection):

  1. Change the media once around 4 or 5dpi with fresh HDF media. The cells are no longer BL2+ after this media change.

Day 12 (7 days post infection):

  1. If assaying proliferation, complete flow cytometry quantification of CellTrace dilution on this day.

  2. If assaying reprogramming rates, re-seed cells on this day.

    1. Gelatin coat a new 96-well plate as usual.

    2. Aspirate the gelatin and coat the plate with laminin (described here).

    3. Dissociate 7dpi cells with 3x diluted trypsin.

    4. Quench with 200 uL HDF media.

    5. Aspirate laminin and transfer dissociated cells to laminin coated plate.

Day 13 (8 days post infection):

  1. 24 hours after replating the cells, media change to N3 media:

    1. N3 media = N3 base + BDNF/CNTF/GDNF (1,000X, 10 µg/mL) + FGF (10,000X, 100 µg/mL)

    2. Spike in 1,000X RepSox to N3 media for all experimental conditions. This differs from MEF reprogramming.

Days 14-33 (9-28 days post-infection):

  1. Do half N3 media changes every 3-4 days (2 times a week). Aspirate ~45 µL media, and add back ~50 µL fresh N3 media.

Day 33 (28 days post-infection): Assay Reprogramming

  1. For flow staining quantification, dissociate cells with DNAse/Papain, and combine 2 96-wells into 1 eppendorf tube.

  2. Optional: Do a Live/Dead stain (e.g. Zombie).

    1. Dilute ZombieNIR™ 1:1000x in PBS.

    2. Wash dissociated and pelleted cells with PBS.

    3. Resuspend cells in 100 uL diluted Zombie stain.

    4. Incubate at room temperature for 15-30 minutes in the dark.

    5. Wash with DMEM + 10% FBS.

    6. Proceed to fixation step.

Note

Zombie NIR™ dye is excited by the red laser and has fluorescence emission maximum at 746 nm.

  1. Proceed with plate or flow staining as described here.

Note

BAD has used the following antibodies for adherent cell staining:

  • 1:500 diluted mouse anti-Tuj1 (BioLegend Cat #801201) with 1:500 diluted anti-mouse AF-488.

  • 1:500 diluted rabbit anti-MAP2 (Cell Signaling Technology Cat #4542S) with 1:500 diluted anti-rabbit AF-546 or AF-647.

  • 1:10 diluted mouse anti-Hb9 (DSHB Cat #81.5C10) with 1:500 diluted anti-mouse secondary.