Protocol for Generating Mouse Induced Pluripotent Stem Cells by Lentiviral Transduction

Experiment Summary

Terminally differentiated somatic cells can be reprogrammed into an embryonic stem cell-like state by the forced expression of defined pluripotency-associated transcriptional factors, including Oct4, Sox2, Klf4, c-Myc, Lin28, and Nanog. These so-called induced pluripotent stem (iPS) cells can give rise to any cell type of the body and thus have tremendous potential for many applications in research and regenerative medicine. Herein, we describe (1) a protocol for the generation of iPS cells from mouse embryonic fibroblasts (MEFs) using a doxycycline (Dox)-inducible lentiviral transduction system; (2) the derivation of clonal iPS cell lines; and (3) the characterization of the pluripotent potential of iPS cell lines using alkaline phosphatase staining, flow cytometry, and the teratoma formation assays.

Materials and Methods

Mouse Embryonic Fibroblasts

1. Mouse embryonic fibroblasts (MEFs), isolated from embryos of any genetic background of interest, can be used for reprogramming experiments.

Reagents for Lentiviral Transduction

Generation and titer determination of lentiviral particles harboring the OKSM construct (OKSM plasmid and the m2rtTA (Ef1a-rtTA-GFP plasmid) construct are described in detail in a preceding chapter in this volume.
Polybrene infection/transfection reagent 10 mg/mL stock (used at 1:1700 dilution).

General Cell Culture Reagents

MEF culture medium (MEF media)
mESC/iPSC culture medium (iPSC media)
Dulbecco's phosphate-buffered saline (DPBS) without calcium and magnesium.
0.1% gelatin solution (w/v) is prepared by mixing 1 g of gelatin from porcine skin with 1 L ultrapure water (milli-Q). Autoclave to dissolve and sterilise.
Cryopreservation medium: 90% FBS (v/v) and 10% dimethyl sulfoxide (DMSO) (v/v).
0.25% trypsin-EDTA.
Doxycycline hyclate diluted to 2 mg/mL in dH₂O (1000 stock).
Mr. Frosty freezing containers.

Equipment for iPS Cell Colony Isolation

Dissection microscope.
Irradiated mouse embryonic fibroblasts (iMEFs) can be generated in house.
24-well plates.

Reagents and Equipment for Flow Cytometry
Alkaline Phosphatase Assay
Teratoma Formation Assay

Procedure

A. Reprogramming of MEFs into iPS Cells

Freshly derive MEFs, as described previously, or thaw low passage (p0–p2) cryopreserved MEFs for reprogramming experiments.
For thawing of cryopreserved MEFs, quickly transfer a cryovial of MEFs (~2–3 million cells) from liquid nitrogen into a 37°C water bath.
Once thawed, quickly transfer MEFs with a pipette into a 15 mL centrifuge tube containing 10 mL of pre-warmed MEF media.
Pellet the cells by centrifugation at 450 g for 3 min.
Remove the supernatant, resuspend the cell pellet (1.5-3 x 10⁶ cells on average) in 12 mL MEF media, and transfer to a T75 cell culture flask with a vented cap to allow the cells to recover for 1-2 days before starting the reprogramming experiments.
One to two days after recovery, cellularize thawed or freshly derived cells as follows: remove MEF media, wash cells once with DPBS to remove traces of serum, and then add 3 mL of 0.25% trypsin-EDTA solution and incubate at 37°C for 3-5 min. Neutralize the enzymatic reaction by the addition of 3 mL MEF media, and pipette medium onto the surface of the flask 3-5 times to dissociate the MEFs. Transfer the cell suspension into a 15 mL tube.
Perform cell counting using a hemocytometer or automated cell counters.
It is recommended to seed cells at a range of 0.5-2.5 x 10³ cells/cm² in gelatin-coated 6-well plates containing MEF media. (Fig. 1)

Fig. 1 Schematic depicting MEF to iPSC reprogramming protocol

9. Twenty-four hours later, perform lentiviral transduction as follows: prepare viral mix by adding polybrene (1:1700), lentivirus-m2rtTA (mean occurance of infection [MOI] of 2), and lentivirus-OKSM (MOI of 2) in 2 mL iPSC media; following this aspirate culture media from wells to be infected, and replace with the iPSC media containing the viral mix (Fig. 1).

10. Perform spin inoculation by transferring the plate(s) into a centrifuge, and spin for 60 min at 750 g at room temperature. Afterward, transfer the plate(s) into a 37°C incubator with 20% O₂ and 5% CO₂.

11. On the next day, remove virus-containing media, and replace with fresh iPSC media supplemented with doxycycline (2 μg/mL) to initiate the reprogramming process (3 mL of media per well of 6-well plates).

12. Perform media changes every other day using doxycycline supplemented iPSC media for the first 6 days of reprogramming.

13. After 6 days, daily media changes are recommended due to increased cell densities. Alternatively, add 6 mL of media into one well of a 6-well plate if media changes can only be performed every other day.

14. Expected changes in cell morphology during reprogramming are shown in Fig. 2. iPS cell colonies should be identifiable after approximately 12 days, and it is recommended to transfer the cells into doxycycline-free iPSC media for another 4 days to remove aberrant iPS cell colonies that are still dependent on forced transgene expression. After this period proceed to isolate clonal lines by colony picking.

Fig. 2 Timeline of reprogramming from MEFs to iPSCs.

B. Isolation of Clonal iPS Cell Lines by Colony Picking

Six hours to 1 day before colony isolation, prepare recipient plates by seeding iMEFs onto gelatin-coated 24-well plates at a density of 2*10⁴ cells/cm2 in 1 mL of iPSC media per well.
Rinse the 6-well plate(s) containing the reprogrammed cultures with DPBS, and then add 1 mL of warm DPBS into each well. Colonies can be picked under an inverted light microscope or a dissection microscope.
Identify a reasonably isolated (i.e., not fused to other colonies) iPS cell colony with characteristically dome-shaped morphology (Fig. 3a(i)).
Using a 20 μL pipette, draw a circle around the colony with a sterile tip to detach the colony from surrounding fibroblasts (Fig. 3a(ii)).
Nudge the colony with the tip to gently lift it from the underlying tissue culture plastic (Fig. 3a(iii)).
Aspirate the free-floating colony with the pipette in a 12.5 μL volume, and transfer into a 1.5 mL tube containing 50 μL of 0.25% trypsin-EDTA (Fig. 3a(iv)).
After 2–4 min, gently further dissociate the transferred colony by pipetting the medium within the tube several times.
Transfer the cell suspension from the tube directly into a well of the prepared 24-well plate with the iMEFs (Fig. 3a(iv)).
Repeat steps 3–8 with other colonies to generate more potential clonal lines.
Change media with fresh iPSC media 24 h after colony picking.
New, dome-shaped colonies should form in the recipient 24-wells after 2 days (Fig. 3a).
Expand new clones through routine passaging to propagate the iPS cells (Fig. 3b) (see Notes 10, 11 and 12) as described for mouse embryonics stem cells in a preceding chapter in this volume.

Fig. 3 Establishment of clonal iPSC lines.

C. Characterization of Clonal iPS Cell Lines

Flow cytometry
Alkaline phosphatase assay
Teratoma formation assay

* For research use only. Not intended for any clinical use.

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Protocol for Generating Mouse Induced Pluripotent Stem Cells by Lentiviral Transduction

Experiment Summary

Materials and Methods

Procedure

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