Protocol | Published:

Generation of mouse-induced pluripotent stem cells with plasmid vectors

Nature Protocols volume 5, pages 418428 (2010) | Download Citation

Abstract

Reprogramming of somatic cells into pluripotent stem cells has been reported by introducing a combination of several transcription factors (Oct3/4, Sox2, Klf4 and c-Myc). The induced pluripotent stem (iPS) cells from patient's somatic cells could be a useful source for drug discovery and cell transplantation therapies. However, to date, most iPS cells were made using viral vectors, such as retroviruses and lentiviruses. Here we describe an alternative method to generate iPS cells from mouse embryonic fibroblasts (MEFs) by continual transfection of plasmid vectors. This protocol takes around 2 months to complete, from MEF isolation to iPS cell establishment. Although the reprogramming efficiency of this protocol is still low, the established iPS cells are most likely free from plasmid integration. This virus-free technique reduces the safety concern for iPS cell generation and application, and provides a source of cells for the investigation of the mechanisms underlying reprogramming and pluripotency.

Introduction

Induced pluripotent stem (iPS) cells can be generated from somatic cells by addition of several combinations of transcription factors (Oct3/4, Sox2, Klf4 and c-Myc) and chemical compounds in mouse, rat, pig, monkey and human1,2,3,4,5,6,7. These iPS cells could supply patient-specific pluripotent stem cells for use in the study of disease pathogenesis, drug discovery, toxicology and cell transplantation therapy. Mouse iPS cells can differentiate into all three germ layers and contribute to chimeric mice after injected into blastocysts8,9,10. Human iPS cells have been shown to differentiate into neurons and cardiomyocytes in vitro 2. However, the original method of iPS generation uses retrovirus vectors that integrate the reprogramming factors into the host genome1. One of the factors, c-Myc, is known as a proto-oncogene, and its reactivation could give rise to transgene-derived tumor formation9. To overcome the safety concern of iPS cell generation, several protocols have been reported, with the omission of c-Myc from the reprogramming factors, removal of the integrated factors after iPS establishment, transient expression of the reprogramming factors using adenovirus vectors or plasmids and direct delivery of reprogramming proteins11,12,13,14,15,16,17,18,19. Current methods for iPS induction can be divided into three categories based on their vector types: virus, DNA and cell penetrating peptide (Table 1). Although retroviral and lentiviral vectors have high reprogramming efficiency, they alter endogenous genomic construction and may increase tumor formation risk. On the other hand, the transient expression methods, such as adenovirus, plasmid and cell penetrating protein, are of low efficient but could avoid genomic alteration of iPS cells. Nevertheless, it should be carefully examined whether there is any difference in the characteristics between iPS cells established by different methods before use in medical applications. For example, when iPS cells are established using retroviral vectors, their tissue of origin affects their differentiation potential20. Retrovirus-derived iPS cells established from mouse embryonic fibroblasts (MEFs) are pluripotent, as they can contribute to adult chimera mice and the germ line at high frequency9. Retrovirus-derived iPS cells from adult tail fibroblasts and hepatocytes can also contribute to chimeric mice21. However, when the iPS cells from tail fibroblasts and hepatocytes are induced to differentiate in vitro into neural cells, these cells frequently form teratomas following cell transplantation. On the other hand, the iPS cells prepared from MEFs showed low teratoma formation similar to embryonic stem (ES) cells. Therefore, comparison of iPS cells by different methods should be done with iPS cells from the same tissue origin. It is also important to assess the tumorigenicity or any abnormal behavior of the cells after in vivo and in vitro differentiation using experimental animal models. As retrovirus-derived iPS cells from MEFs showed high differential potential, MEFs would be a suitable source to find any negative effect of the establishing methods on iPS cell potential.

Table 1: iPS induction methods.

We have recently shown mouse iPS cell generation by transient expression of the reprogramming factors using plasmid vectors19. Here we describe the protocol in detail. We designed two plasmids: one encoding Oct3/4, Klf4 and Sox2 and the other encoding c-Myc (Fig. 1a). The former three transcription factors are connected with a 2A self-cleaving peptide from the foot-and-mouth disease virus, which enables polycistronic expression of these proteins from one coding region22,23. Repeated transfection of the plasmids can induce pluripotent stem cells from MEFs (summarized in Fig. 1b). This protocol includes MEF isolation, feeder preparation, iPS induction with plasmids and PCR screening to obtain iPS cells without evidence of genomic integration. Although we cannot rule out the integration of short plasmid fragments completely, we can at least show that the resulting iPS cells do not retain functional transgene expression. The plasmid-iPS cells can develop teratomas when subcutaneously injected into nude mice and contribute to adult chimeras.

Figure 1: Schematic diagram of iPS cell generation with plasmid vectors.
Figure 1

(a) Expression vectors for iPS cell generation. pCX-OKS-2A (left) encodes three transcription factors (Oct3/4, Sox2 and Klf4) connected with 2A self-cleaving peptide (2A). The locations of CAG promoter (CAG), stop codon (stop) and polyadenylation signal (pA) are also indicated. From this vector, three separate proteins are produced. The c-Myc expression vector (pCX-cMyc) is shown on the right. (b) Approximate time table of the iPS cell generation. Shown are the procedures and the corresponding step numbers. Cell culture medium is also described below.

This protocol provides virus-free iPS cells that are helpful for elucidating the mechanisms underlying reprogramming and pluripotency. The cells are available to find the difference between mouse iPS cells and ES cells under transgene-free condition. It is also possible to examine the effect of induction method on the characteristics of iPS cells by comparing iPS cells derived from plasmid and other methods.

Limitations of the protocol

When using this protocol, infectious viral vectors are not required to generate iPS cells. However, the established rate of plasmid-iPS cells using this plasmid-based protocol is much lower than that of viral induction, and this method was only applied to MEF, many improvements would be required to induce iPS cells from other tissue origins or other species. For example, the induction efficiency of iPS cells from human fibroblasts is very low and requires longer expression periods of reprogramming factors than mouse fibroblasts24. The reprogramming efficiency could be increased by incorporating other reprogramming factors (e.g., Lin28, Nanog, Esrrb, UTF1 and suppression of the p53 pathway)25,26,27,28, modification of cell signaling (Wnt)29 and treatment with chemical drugs (valproic acid and 5′-azacytidine)30. The episomal plasmid vector is one solution to achieve high and long-term expression of reprogramming factors. With these modifications and future improvement, human iPS cells can be established from adult cells with virus- and integration-free methods. However, the effect of the modifications on the differentiation potential of iPS cells should be examined carefully, and this protocol provides basic methods and iPS cells for the comparison.

Experimental design

Plasmids. The plasmids used in this protocol are pCX-OKS-2A and pCX-cMyc (Fig. 1a, whole sequences are available from Addgene). As plasmids are easily degraded and diluted in culture condition, we gathered the three core transcription factors (Oct3/4, Klf4 and Sox2) into one plasmid. With this modification, the expression balance of these factors is fixed, and one copy of pCX-OKS-2A can express all three transcription factors. Stop codons of Oct3/4 and Klf4 are deleted, and the three factors are connected with a 2A self-cleaving peptide. Only Sox2 keeps its own stop codon. In total, they form one open reading frame, which is expressed from a strong promoter, CAG31. The 2A sequence encodes around 30 amino acid residues that is separated before proline residue at C-terminal by an unknown mechanism. Therefore, proteins (Oct3/4 and Klf4), which are located upper stream of 2A sequence, has around 29 residues of additional amino acids on their C-terminal, whereas downstream proteins (Klf4 and Sox2) have a few additions on N-terminal. These extra amino acids may influence on the function of three transcription factors.

MEF. Figure 1b illustrates the summary of this protocol. It takes almost 2 months from MEF isolation to iPS establishment. We use a reporter mouse, which expresses green fluorescent protein (GFP) and a drug resistant gene under the promoter control of a pluripotent cell marker, Nanog, to generate the MEF. The isolation of the fibroblasts described in this protocol is essentially the same as our previous publication30. The fluorescent protein helps us to find reprogrammed cells easily, and non- or partially reprogrammed cells can be eliminated by the drug selection, because cells that do not express Nanog will not be drug resistant. From one embryo, we can obtain sufficient numbers of MEFs (around 5 × 106) to perform several experiments at passage 1 (Step 9). The age of the starting fibroblast culture (passage number) is also a critical factor for iPS generation. We recommend using MEFs within passage 3 for iPS production to avoid replicative senescence. After transfection of plasmids encoding reprogramming factor, we switch the culture medium for fibroblasts to medium for ES cells on day 4. We just followed our previous protocol for the timing of medium change and do not have any data regarding its effect on reprogramming efficiency; however, an early switch may promote iPS induction. On day 10, MEFs are transferred to feeder cells, which is a favorable culture condition for mouse ES cell. An earlier switch in the culture condition may promote iPS induction efficiency. We use SNL cells as feeder cells after interrupting their proliferation by mitomycin C treatment. The cells are clonally derived from an STO cell line and stably express leukemia inhibitory factor (LIF), which is a strong signal for mouse ES cells to maintain their pluripotent status32. As MEFs themselves also secrete LIF and behave as feeder cells, iPS cell can be established from MEFs without feeder cells. Puromycin selection with Nanog reporter MEF can reduce non-reprogrammed cells and increase GFP-positive population. We retrovirally introduced puromycin-resistant gene into feeder cells and use them during the drug treatment.

Plasmid transfection of MEF. iPS cell induction from MEF require continuous expression of reprogramming factor for around 12 d (ref. 33). After transfection into MEF, the plasmid vectors are gradually degraded and diluted by cell proliferation. Therefore, the protocol includes four rounds of repeated transfection to maintain the transgene expression in MEFs. However, in some cases, iPS cells were established by only two rounds of transfection. The two transfection reagents (FuGENE 6 and FuGENE HD, Roche) we tested could induce iPS cells, but GeneJuice (Novagen) failed to produce iPS cells in this assay. However, when we used GeneJuice we achieved a high-transfection efficiency, but the reagent appeared to be toxic for MEFs. Thus, the balance of transfection efficiency and cell toxicity is important when making iPS cells with plasmid vectors. To monitor the transfection efficiency, it is useful to include control experiment with an expression vector encoding fluorescent protein (e.g., DsRedExpress) in the experiment. We used this protein to show a routine transfection efficiency of more than 40% on day 10 (Step 36, Fig. 2). It also serves as a negative control for iPS cell induction, as ES-like colonies do not emerge without reprogramming factors. The quality of plasmid vectors are also important when making integration free-iPS cells. We use an anion-exchange-based column (QIAGEN plasmid kit; QIAGEN) to purify the plasmids. To avoid nicking and degradation, the plasmids should be aliquoted and stored at −20 °C.

Figure 2: Transfection efficiency of repeated lipofection.
Figure 2

Expression plasmid encoding DsRedExpress was transfected four times with FuGENE 6 reagent. Their phase images (left) and fluorescence (middle) were photographed on day 10 of the iPS induction and examined by flow cytometory (right). The numbers indicate the percentages of fluorescence positive cell populations. MEFs were also infected with pMXs-based retrovirus encoding DsRedExpress on day 2 and were analyzed. Scale bar, 500 μm.

Picking colonies. ES-like colonies first become visible approximately on day 20 after post-transfection with a tightly packed dome-like structure. They should become large enough to be picked up on around day 30 (Fig. 3). When MEFs from Nanog reporter mice, the fluorescence will aid in picking superior colonies. However, we found that some GFP-negative or colonies with weak fluorescence become GFP-positive after being picked up. ES-like colonies are mechanically selected and transferred to trypsin solution prepared in a 96-well plate. This step should be finished within 15 min, as the trypsin solution is concentrated due to evaporation. After digestion, the cells are transferred into a 24-well plate. We refer to this point as passage 1. If the colonies are large enough, one can expand half of them for proliferation and the other half for PCR screening of genomic integration (described below). We have previously described the picking of iPS colonies and their further expansion34. To take gross images of the culture dish, we fix cells with methanol and stain with crystal violet34.

Figure 3: Morphology of iPS colonies just before picking up.
Figure 3

The phase images (upper) and fluorescence (lower) of typical ES-like colonies on day 24 after the first transfection. Lower panels showed GFP fluorescence from NanogGFP-IRES-Puro locus. Although the right colony barely showed the fluorescence, it became GFP-positive after picking up. Scale bar, 500 μm.

In some experiments (10%), more than 500 colonies appear. This may be because the transfected plasmid(s) have integrated into the fibroblast's genome and transformed the cells to non-ES-like cells in the early period of the iPS cell induction. After several rounds of proliferation, these cells spread out across the 100-mm dish by the passage on day 10. In these cases, we failed to obtain iPS clones without apparent integration. As nicked plasmid tends to integrate into genome, the use of high quality of plasmid is important. We recommend carrying out several independent experiments at the same time.

Detection of genomic plasmid integration. To select iPS colonies for further cultivation, genomic integration of plasmids is evaluated by PCR. A cell lysate is prepared from a portion of ES-like cells grown on a 24-well plate and is used for screening with the primer sets for each plasmid. To detect pCX-OKS-2A, we use the primers for Klf4 (Fig. 4a). The primers are located on two independent exons flanking one intron. Therefore, they amplify an 831-bp fragment from the endogenous locus and 186 bp from the transgene (Fig. 4b). The endogenous amplicon functions as a positive PCR control. For pCX-cMyc, we use the primers for c-Myc, which amplify a 541-bp fragment from the endogenous locus and 237 bp from the transgene (Fig. 4a,b).

Figure 4: PCR screening of genomic integration.
Figure 4

(a) Schematic diagram of PCR primers. Open boxes indicate exons of Klf4 (left) and c-Myc (right), and arrows indicate primer sites. The primers of Klf4 are located on two exons flanking intron. Therefore, they amplify 831 bp from endogenous locus and 186 bp from transgene. The primers for c-Myc amplify 541 bp from endogenous locus and 237 bp from transgene. (b) Detection of genomic integration by PCR. Open and black arrowheads indicate bands from endogenous alleles and transgenes, respectively. Retrovirus-induced iPS cells (retro-iPS) were used as a control. Some clones (nos. 2, 3 and 8) do not seem to have integration. Clone 8 showed smaller band than expected size with Klf4 primers. That is most likely derived from primer dimers.

iPS cell freezing. After the iPS cell expansion, we make large amount of freeze stocks of iPS clones at early passages30. These stocks should be stored in the vapor phase of liquid nitrogen. The recovery of iPS cells after freezing is 50%. The established iPS cells are ready for further characterization, such as gene expression profile, detailed genomic integration, teratoma formation and chimera mice contribution (Fig. 5)19.

Figure 5: Pluripotency of plasmid-iPS cells.
Figure 5

Plasmid-iPS cells developed teratoma when subcutaneously transplanted into nude mice (ad). (a) Hematoxylin and eosin staining sections of neural tissue, (b) gut-like epithelial tissue, (c) epidermal tissue and (d) striated muscle. Scale bar, 50 μm. (e) The iPS cells also contributed to chimeric mouse. iPS cells gave rise to gray hair. These experiments were performed according to Animal Research Committee in Kyoto University.

Materials

Reagents

  • pCX-OKS-2A (Addgene, plasmid no. 19771)

  • pCX-cMyc (Addgene, plasmid no. 19772)

  • Nanog GFP-IRES-Puro mice 9 (Riken Bioresource Center, BRC no. RBRC02290)

  • SNL feeder cells35 (Health Protection Agency Culture Collections)

  • DMEM containing 4.5 g l−1 glucose (Nacalai Tesque, cat. no. 08459-35)

  • Fetal bovine serum (FBS; Invitrogen, cat. no. 26140-079)

  • PBS without calcium and magnesium (Nacalai Tesque, cat. no. 14249-95)

  • l-Gln (Invitrogen, cat. no. 25030-081)

  • Non-essential amino acid solution (Invitrogen, cat. no. 11140-050)

  • 2-Mercaptoethanol (Invitrogen, cat. no. 21985-023)

  • Penicillin/streptomycin (Invitrogen, cat. no. 15140-122)

  • 0.25% (wt/vol) Trypsin/1 mM EDTA solution (Invitrogen, cat. no. 25200-056)

  • Gelatin (Sigma, cat. no. G1890) (see REAGENT SETUP)

  • Dimethylsulfoxide (DMSO; Sigma, cat. no. D2650)

  • Puromycin (Sigma, cat. no. P7255) (see REAGENT SETUP)

  • Opti-MEM I Reduced-Serum Medium (Invitrogen, cat. no. 31985-062)

  • FuGENE 6 Transfection Reagent (Roche, cat. no. 11 814 443 001)

  • ES medium (see REAGENT SETUP)

  • SNL medium (see REAGENT SETUP)

  • FP medium (see REAGENT SETUP)

  • Proteinase K (Nacalai Tesque, cat. no. 29442-14) (see REAGENT SETUP)

  • TaKaRa Ex Taq (Takara Bio, cat. no. RR001A)

  • Cell lysis buffer (see REAGENT SETUP)


Equipment

  • 100-mm tissue culture dish (Falcon, cat. no. 353003)

  • Six-well tissue culture plate (Falcon, cat. no. 353046)

  • 24-well tissue culture plate (Falcon, cat. no. 353047)

  • 96-well tissue culture plate (Falcon, cat. no. 351172)

  • 15-ml conical tube (Falcon, cat. no. 352196)

  • 50-ml conical tube (Falcon, cat. no. 352070)

  • 1-ml plastic disposable pipette (Falcon, cat. no. 357520)

  • 5-ml plastic disposable pipette (Falcon, cat. no. 357543)

  • 10-ml plastic disposable pipette (Falcon, cat. no. 357551)

  • 25-ml plastic disposable pipette (Falcon, cat. no. 357525)

  • Bottle-top filter (Techno Plastic Products, cat. no. 99500)

  • 0.22-μm pore size filter (Millex GP; Millipore, cat. no. SLGP033RS)

  • 10-ml disposable syringe (Terumo, cat. no. SS-10ESZ)

  • Dissecting forceps

  • Dissecting scissors

  • Coulter counter (Z2; Beckman Coulter)

  • 0.2-ml PCR tube (Greiner Bio-One, cat. no. 301301)

  • Freezing container (Nalgene, cat. no. 5100-0001)


Reagent Setup

Gelatin stock solution (10×)

  • Dissolve 1 g of gelatin powder in 100 ml of distilled water, autoclave and store at 4 °C for 2 months.

Gelatin solution (1×)

  • Warm the 10× gelatin stock to 37 °C, add 50 ml of the stock to 450 ml of distilled water. Filter the solution with a bottle-top filter (0.22 μm) and store at 4 °C for 2 weeks.

Gelatin-coated culture dishes

  • Add a sufficient volume of 1× gelatin solution to cover the area of a culture dish. For example, 1, 3 or 5 ml of gelatin solution is used for a 35-, 60- or 100-mm dish, respectively. Incubate the dish for at least half an hour and not longer than 4 h at 37 °C. Before using, aspirate excess gelatin solution.

Culture dishes covered with feeder cells

  • Seed feeder cells on gelatin-coated culture dishes as described previously34. Use within 2 weeks.

Puromycin (10 mg ml−1)

  • Dissolve puromycin in distilled water to a final concentration of 10 mg ml−1. Filter-sterilize through a 0.22-μm filter. Aliquot and store at −20 °C for 3 months.

FP medium

  • DMEM containing 10% FBS (vol/vol), and 50 U and 50 mg ml−1 penicillin and streptomycin, respectively. To prepare 500 ml of FP medium, mix 50 ml of FBS and 2.5 ml of penicillin/streptomycin, and then fill to 500 ml with DMEM. Store at 4 °C for a week.

ES medium

  • DMEM containing 15% FBS (vol/vol), 2 mM l-Gln, 1 × 10−4 M non-essential amino acids, 1 × 10−4 M 2-mercaptoethanol, and 50 U and 50 mg ml−1 penicillin and streptomycin. To prepare 500 ml of the medium, mix 75 ml of FBS, 5 ml of l-Gln, 5 ml of nonessential amino acids, 1 ml of 2-mercaptoethanol and 2.5 ml of penicillin/streptomycin, and then fill to 500 ml with DMEM. Store at 4 °C for a week.

2× Freezing medium

  • To prepare 10 ml of 2× freezing medium, mix 2 ml of DMSO, 2 ml of FBS and 6 ml of DMEM, and sterilize through a 0.22-μm filter. Prepare freshly before freezing cell.

Proteinase K

  • Dissolve Proteinase K in distilled water at 10 mg ml−1. Aliquot and store at −20 °C for 3 months.

Cell lysis buffer

  • PCR buffer containing 150 μg ml−1 of Proteinase K. To prepare 100 μl of cell lysis buffer, mix 10 μl of 10× Ex Taq buffer containing magnesium, 1.5 μl of 10 mg ml−1 Proteinase K and 88.5 μl of distilled water. Prepare freshly before use.

PCR solution

  • To set up 20 μl of PCR solution, mix 2 μl of 10× Ex Taq buffer containing magnesium, 1.6 μl of 2.5 mM of dNTP mix, 0.25 μl of 20 μM primers, 0.1 μl of Ex Taq polymerase and 1 μl of template, and distilled water. Keep it on ice and use immediately.

Procedure

Preparation of fibroblasts from mouse embryos

Timing: 15 d

  1. Kill a 13.5-d pregnant female mice by cervical dislocation. Detection of the vaginal plug is designated as 0.5-d post coitum. Isolate the uteri and wash with 20 ml of PBS briefly at room temperature (20–25 °C)36.

  2. Separate the embryos from the placenta and surrounding membranes using forceps. Remove the head, visceral tissues and gonads from the isolated embryos.

  3. Transfer the embryos to a 100-mm dish containing 1 ml of room temperature PBS. Mince the bodies until it becomes around 2 mm length using a pair of scissors, transfer into a 50-ml conical tube containing 0.25% (wt/vol) trypsin/1 mM EDTA solution (3 ml per embryo) and incubate at 37 °C for 20 min.

  4. Add an additional 3 ml of 0.25% (wt/vol) trypsin/1 mM EDTA solution per embryo and incubate the mixture at 37 °C for 20 min.

  5. Add 6 ml per embryo of FP medium, and pipette up and down a few times to aid tissue dissociation.

  6. Incubate the mixture without agitation for 5 min at room temperature to remove debris and transfer the supernatant into a new 50-ml conical tube. Centrifuge at 200g for 5 min at room temperature, discard the supernatant and resuspend the pellet in 6 ml per embryo of FP medium.

  7. Count the cell number and adjust the concentration to 1 × 106 cells per ml with FP medium. Generally, 1 × 107 cells can be obtained from a single embryo. Transfer the cell suspension to 100-mm tissue culture dishes (1 × 107 cells per dish) and incubate at 37 °C with 5% CO2 for 24 h (passage 1).

  8. Remove the medium (and floating cells) and wash with 10 ml of PBS. Add 10 ml of new FP medium.

  9. When the cells become confluent (generally 2 or 3 d after the isolation), remove the FP medium and wash the cell monolayer with 10 ml of PBS.

  10. Remove PBS and add 1 ml of 0.25% (wt/vol) trypsin/1 mM EDTA and incubate for 5 min at 37 °C.

  11. Once the cells have detached from the dish, add 9 ml of FP medium and resuspend by pipetting. Usually we obtain 5 × 106 cells from one 100-mm dish.

  12. At this step, the cells can be either passaged using option A or frozen using option B.

    1. Passaging of MEFs

      1. Dilute the cells 1:4 and divide into four new 100-mm dishes (passage 2).

        Critical step

        • For the generation of iPS cells, we use MEFs within three passages to avoid replicative senescence.

      Timing: 15 min

    2. Preparation of freeze stock of MEF

      1. Transfer the cell suspension to a 15-ml tube, count the cell number and spin the cells at 160g for 5 min at room temperature.

      2. Discard the supernatant and resuspend the cells with FP medium to a concentration of 1 × 107 cells per ml.

      3. Prepare 2× freezing medium (see REAGENT SETUP) and aliquot 0.5 ml into freezing vials.

      4. Transfer 0.5 ml of the cell suspension to the freezing vials and mix gently by pipetting.

      5. Place the vials in a cell-freezing container and keep at −80 °C overnight.

        Pause point

        • For long-term storage, keep frozen cells in the gas phase of a liquid nitrogen tank.

      Timing: 1 h


iPS induction with plasmid vector: preparation of fibroblasts (day 1)

Timing: 1 h

  1. Prepare 9 ml of FP medium in a 15-ml tube.

  2. Remove a vial of frozen MEFs from the liquid nitrogen tank and put the vial into 37 °C water bath until most (but not all) cells are thawed.

  3. Wipe the vial with ethanol, open the cap and transfer the cell suspension to the tube prepared in Step 13.

  4. Centrifuge at 160g for 5 min at room temperature and then discard the supernatant.

  5. Resuspend the cells with 10 ml of FP medium and transfer to a gelatin-coated 100-mm dish. Incubate the cells in a 37 °C, 5% CO2 incubator, until the cells become 80–90% confluent (2 × 106 cells per dish). Aspirate the medium and wash the cells with 5 ml of PBS.

  6. Remove PBS completely, add 1 ml of 0.25% (wt/vol) trypsin/1 mM EDTA and incubate at 37 °C for 5 min.

  7. Add 9 ml of the FP medium and suspend the cells by pipetting up and down to single-cell suspension.

  8. Wash with PBS and trypsinize the cells as described in Steps 9–11. Transfer the cells to a 50-ml tube and count the cell numbers. Adjust the concentration to 6.5 × 104 cells per ml. Transfer 2 ml of cell suspension (1.3 × 105 cells) to each well of a six-well plate. Prepare one additional well for control experiment. Incubate the dish overnight at 37 °C, 5% CO2.


iPS induction with plasmid vector: Plasmid transfection (day 2)

Timing: 1 h

  1. Aspirate the medium from a fibroblast dish and add 2 ml of fresh FP medium.

  2. Transfer 0.1 ml of Opti-MEM into a 1.5-ml tube.

  3. Transfer 4.5 μl of FuGENE 6 Transfection Reagent directly into the Opti-MEM prepared in Step 22, mix gently by finger tapping and incubate for 5 min at room temperature.

  4. Mix 1.0 μg of pCX-OKS-2A and 0.5 μg of pCX-cMyc, and add them to the FuGENE 6/Opti-MEM containing tube, mix gently by finger tapping and incubate for 15 min. For control experiment, use appropriate plasmid encoding fluorescent protein instead of the above two plasmids. We use expression plasmid encoding red fluorescent protein, DsRedExpress, to monitor the transfection efficiency.

  5. Add the DNA/FuGENE 6 complex dropwise into the fibroblast dish and incubate overnight at 37 °C, 5% CO2.


iPS induction with plasmid vector: medium change (day 3)

Timing: 5 min

  1. Aspirate the medium from a fibroblast dish and add 2 ml of fresh FP medium.


iPS induction with plasmid vector: plasmid transfection (day 4)

Timing: 1 h

  1. Repeat Steps 21–25.


iPS induction with plasmid vector: medium change (day 5)

Timing: 5 min

  1. Repeat Step 26.


iPS induction with plasmid vector: plasmid transfection (day 6)

Timing: 1 h

  1. Repeat Steps 21–25 except that ES medium is used instead of FP medium.


iPS induction with plasmid vector: medium change (day 7)

Timing: 5 min

  1. Repeat Step 26 but add 2 ml of fresh ES medium.


iPS induction with plasmid vector: plasmid transfection (day 8)

Timing: 1 h

  1. Repeat Steps 21–25 using 2 ml of ES medium instead of FP medium.


iPS induction with plasmid vector: medium change (day 9)

Timing: 5 min

  1. Repeat Step 30.


iPS induction with plasmid vector: passage of fibroblast (day 10)

Timing: 1 h

  1. Discard the medium and wash the cells once with 2 ml of PBS.

  2. Aspirate the PBS, and add 0.5 ml per well of 0.25% (wt/vol) trypsin/1 mM EDTA and incubate for 5 min at 37 °C.

  3. Add 4.5 ml of ES medium, and break up the cells into a single-cell suspension by pipetting up and down several times.

  4. Count the cell concentration and adjust to 1 × 105 cells per ml with ES medium. Cell number on day 10 is 1.2–1.7 × 106. Transfer 10 ml of the suspension (1 × 106 cells) to a 100-mm dish covered with feeder cells34. Incubate the cells at 37 °C, 5% CO2. At this point, the transfection efficiency should be examined with the control transduced cells. They are harvested by trypsinization as in Steps 33–35 and then analyzed by flow cytometer.

    Critical step

    • It is possible to use gelatin-coated dishes; however, they tend to reduce the induction efficiency.


iPS induction with plasmid vector: medium change (day 11–30)

Timing: 5 min each day

  1. Change the medium every other day until the colonies grow large enough (0.5–1.0 mm diameter) to be picked up (Fig. 3).

    Troubleshooting


Picking up the iPS colonies

Timing: 1 h

  1. Add 20 μl of 0.25% (wt/vol) trypsin/1 mM EDTA to each well of a 96-well plate.

  2. Remove the medium from the cells from Step 37 and add 10 ml of PBS to each dish.

  3. Aspirate the PBS and add an additional 5 ml of PBS.

  4. Pick a colony from the dish using a Pipetman set at 2 μl and transfer it into the 96-well trypsin plate (Step 38). Pick up as many colonies as possible within 15 min. Incubate for a further 15 min in trypsin at 37 °C to dissociate into single cells.

    Critical step

    • Pick for a maximum of 15 min as the trypsin solution will become more concentrated due to evaporation.

  5. Add 180 μl of ES medium to each well, and pipette up and down to break up the colony to single cells.

  6. Transfer the cell suspension from one well of the 96-well plate into a well of 24-well plates containing feeder cells, add 300 μl of ES medium and incubate in 37 °C, 5% CO2 incubator until the cells reach 50–60% confluence. When using NanogGFP-IRES-Puro MEFs, treatment of puromycin (final concentration; 1.5 μg ml−1) support to select highly reprogrammed cells and decrease non-reprogrammed MEF cells. At this point, they could be screened by genomic PCR to exclude clones with apparent integrations (see Steps 44–53). After colony picking up (Step 41), the remaining colonies in a 100-mm dish can be stained with crystal violet to visualize the gross image30.

    Troubleshooting


Examination of genomic integration

Timing: 1 d

  1. Aliquot 5 μl of cell lysis buffer into 0.2-ml PCR tubes.

  2. Aspirate the medium and wash the cells in 24-well plates with 0.5 ml of PBS.

  3. Remove the PBS and add an additional 0.5 ml of PBS.

  4. Harvest a part of cells (0.1 μl of volume) by scraping from the dish using a Pipetman set at 2 μl and transfer it into the cell lysis buffer.

  5. After picking the colonies, aspirate PBS completely from culture dishes and then add 0.5 ml of ES medium, and return the cultures to the 37 °C, 5% CO2 incubator until the cells reach 80–90% confluence.

  6. Incubate cell lysis buffer at 55 °C for 4–12 h in a humidified chamber.

  7. Add 10 μl of distilled water and incubate at 95 °C for 3 min to inactivate proteinase K.

  8. Use 1 μl of the solution as a template for 20 μl of PCR solution. Primer sequences and PCR conditions are listed in Table 2. Keep the mixture on ice.

  9. Run the PCR using a thermal cycler.

  10. Separate PCR products with agarose gel electrophoresis. Select the clones that show bands from endogenous allele only for further cultivation (Fig. 4).

    Troubleshooting

Table 2: PCR conditions.

Expansion of iPS cells

Timing: 1 h

  1. Aspirate the medium and wash the cells with 1 ml of PBS.

  2. Remove the PBS completely, add 0.1 ml of 0.25% (wt/vol) trypsin/1 mM EDTA and incubate at 37 °C for 10 min.

  3. Add 0.4 ml of the ES medium and suspend the cells by pipetting up and down to form a single-cell suspension.

  4. Transfer the cell suspension to a well of six-well plate containing SNL feeder cells (use puromycin-resistant feeder cells for Nanog GFP-IRES-Puro), add 1.5 ml of ES cell medium and incubate in a 37 °C, 5% CO2 incubator. Addition of puromycin (1.5 μg ml−1) supports to select highly reprogrammed cells. When the cells reach 80–90% confluence (usually it occurs within a week), prepare frozen stock of the cells as previously described30.

    Troubleshooting

Troubleshooting

Troubleshooting advice can be found in Table 3.

Table 3: Troubleshooting table.

Timing

Steps 1–12, preparation of fibroblasts from mouse embryos: 15 d

Steps 13–37, iPS induction with plasmid vectors: 30 d

Steps 38–43, picking up the iPS colonies: 1 h

Steps 44–53, examination of genomic integration: 1 d

Steps 54–57, expansion of iPS cells: 1 h

Anticipated results

During the series of plasmid transfection, some floating cells appear because of the cytotoxicity of repeated transfection. However, other cells grow robustly and become confluent around day 10. The transfection efficiency can be evaluated by analyzing control transfected cells using a flow cytometer (Fig. 2). The rate of fluorescent positive cells is regarded as the transfection efficiency. More than 40% can be attained if the procedure goes well.

We obtained iPS colonies without evidence of integration from six out of ten experiments, but not from four experiments including one experiment showing no iPS colony formation. The number of colonies in a 100-mm dish is generally small (below 100). On average, only two plasmid-iPS colonies without evidence of integration appeared from 1 × 106 cells seeded on feeder cells. The estimated established rate is <0.0002%, which is at least 1,000-fold lower than that of viral induction19. Although iPS colonies with genomic integration were frequently observed, genomic PCR screening (Steps 44–53, Fig. 4) can distinguish them easily. The bands from transgene allele indicate genomic integration of the clones. When the PCR products show only bands from endogenous alleles in both primer sets, the iPS clone is a candidate for non-genomic integration clone. We found that almost one-third of ES-like colonies did not show transgene-derived band in this screening. Using Nanog GFP-IRES-Puro MEF, GFP-positive cells became first apparent around day 19 after the induction (Fig. 3). Some clones did not show fluorescence at the time of colony picking up. However, they became GFP-positive during the culture in a 24-well plate.

After picking up and PCR selection, most clones show ES-like proliferation and morphology, including a round-shaped, large nucleoli and scant cytoplasm, in ES cell culture condition. However, some clones show non-ES-like morphology or fail to proliferate. Most clones express ES cell marker gene, such as Nanog, ERas, Zfp42 and Utf1 30. When subcutaneously injected into immuno-deficient mice, most iPS clones develop teratoma containing several tissues, such as neural tissue, gut-like epithelial tissue, epidermal tissue and striated muscle (Fig. 5a–d)19. The differentiation ability into three germ layers shows pluripotency of the cells. Figure 5e indicates adult chimeric mouse derived from the plasmid-iPS clone19. We transplanted the iPS cells into blastocyst of ICR mouse. White and gray hair of the mouse shows contribution of the host and the iPS cells, respectively. These data proved pluripotency of the cells.

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Acknowledgements

We are grateful to Drs. M. Nakagawa, K. Yae, M. Koyanagi and K. Tanabe for scientific discussion, and to K. Takeda and T. Ishii for critical reading of the paper. We also thank T. Ichisaka, K. Okuda, M. Narita, A. Okada, N. Takizawa, R. Kato, R. Iyama, E. Nishikawa, Y. Shimazu and N. Maruhashi for technical and administrative supports. We also thank J. Miyazaki for the CAG promoter. This study was supported in part by a grant from the Program for Promotion of Fundamental Studies in Health Sciences of NIBIO, a grant from the Leading Project of MEXT, a grant from Uehara Memorial Foundation and Grants-in-Aid for Scientific Research of JSPS and MEXT (to S.Y.). K.O. was a JSPS research fellow. H.H. is supported by a Japanese Government (MEXT) Scholarship.

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Affiliations

  1. Center for iPS Cell Research and Application (CiRA), Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto, Japan.

    • Keisuke Okita
    • , Kazutoshi Takahashi
    •  & Shinya Yamanaka
  2. Department of Stem Cell Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.

    • Hyenjong Hong
    •  & Shinya Yamanaka
  3. Yamanaka iPS Cell Special Project, Japan Science and Technology Agency, Kawaguchi, Japan.

    • Shinya Yamanaka
  4. Gladstone Institute of Cardiovascular Disease, San Francisco, California, USA.

    • Shinya Yamanaka

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Contributions

K.O. prepared most of the paper with the assistance of H.H., and K.T. and S.Y. provided advice and proofread the paper.

Corresponding author

Correspondence to Keisuke Okita.

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DOI

https://doi.org/10.1038/nprot.2009.231

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