Gammaretroviral vector encoding a fluorescent marker to facilitate detection of reprogrammed human fibroblasts during iPSC generation

Cells

Human embryonic kidney 293T (HEK293T) cells were obtained from American Type Culture Collection (ATTC; catalog number SD-3515) and maintained in Dulbecco’s modified Eagle’s medium containing 2 mM L-glutamine, 100 U/ml of penicillin, 100 µg/ml streptomycin and 10% heat-inactivated fetal bovine serum (FBS) (Hyclone/ThermoFisherScientific, USA). Human lung fibroblasts were obtained from ATCC (MRC-5, catalog number CCL-171) and maintained in Eagle’s minimal essential medium containing 10% FBS. Mouse ES feeder cells (SNL-76/7-4) that express leukemia inhibitory factor (LIF) and puromycin phosphotransferase were obtained from Wellcome Trust Sanger Institute and maintained in Knockout DMEM with 7% FBS, Penicillin (50 U/ml) and Streptomycin (50 µg/ml). The mouse feeder cells are also available from ATCC (catalog number SNLP 76/7-4).

Plasmids

The following plasmid vectors were obtained from Addgene.org: Plasmids pMXs-hOCT3/4 (catalog number 17217), pMXs-hSOX2 (catalog number 17218), pMXs-hKLF4 (catalog number 17219), and pMXs-hc-MYC (catalog number 17220) were made available by the Yamanaka Laboratory. Plasmids pUMVC (encodes Murine leukemia virus Gag/Pol, catalog number 8449), and pCMV-VSV-G (encodes VSV-G envelope, catalog number 8454) were made available by the Weinberg Laboratory. Plasmid pMXs-mRFP1 encodes monomeric red fluorescent protein (Hotta et al., 2009b) (catalog number 21315) and was made available by the Ellis Laboratory.

Production of vector stocks

Vector stocks were produced in HEK293T cells using CaPO₄-mediated transient transfection protocol (Srinivasakumar, 2002). In preparation for transfection, T-75 cell culture flasks were seeded with 7.5 × 10⁶ HEK293T cells on the previous day. The plasmid DNAs (7.5 µg of pUMVC, 0.6 µg of pCMV-VSV-G and 22.5 µg of the gene-transfer vector encoding the RF or mRFP1) were resuspended in 1.5 ml of CaCl₂ solution (0.25 M). The DNA was precipitated by adding drop wise 1.5 ml of HEPES buffered saline, pH 7.05 (50 mM HEPES, 10 mM KCl, 12 mM dextrose, 280 mM NaCl, 1.5 mM Na₂HPO₄), while bubbling air through the DNA-CaCl₂ solution. The mix was immediately distributed drop wise onto the HEK293T cells. The following day, the medium was replaced with fresh medium, and vector-containing supernatant was harvested 48 h later by centrifugation at 1,400 × g for 15 min at 4°C and stored at −80°C in aliquots. After determination of vector titers (see below), pools of required vectors were made to give the desired multiplicity of infection (MOI), and concentrated by ultracentrifugation at 100,000 × g for 2 h at 4°C. The pelleted vectors were resuspended in a minimal volume of complete MRC-5 growth medium for transduction of human fibroblasts (see below).

Determination of vector titers

HEK293T cells were seeded in 6-well plates (2.5 × 10⁵ cells/well) the day prior to infection. The next day, an aliquot of the vector-containing supernatant was added to the cells in one ml of fresh medium containing 8 µg of polybrene. After overnight incubation, polybrene was diluted by adding an additional 2 ml of fresh medium. The following day, the cells were rinsed several times with PBS and released by trypsin-EDTA treatment. The cells were pelleted and washed with PBS prior to isolation of genomic DNA using DNeasy kits from Qiagen (Maryland, USA) using the manufacturer’s recommended protocol and included an RNAse I treatment step.

The vector and β-actin copy numbers in the isolated genomic DNA were determined using qPCR in a Bio-Rad MyiQ thermocycler. Each qPCR reaction was carried out in a 25 µL final volume of iQ SYBR Green Supermix containing 60 ng of genomic DNA and 200 nM of each primer. We used a two-step PCR with a 95°C for 20 s denaturation step and 63.1°C for 45 s annealing and extension step. A final melt-curve analysis was done with 0.5°C temperature increments.

Transduction of human lung fibroblasts

MRC-5 fibroblasts were seeded in 6-well plates (60,000 cells/well). The cells were transduced on two consecutive days with pooled vector stocks containing all four RF vectors and the mRFP1 vector at an MOI of 5 using the “spin-transduction” method. This was done by centrifuging the plates at 1,200 × g for 2 h at room temperature (24°C) after the addition of the vectors in 1 ml of MRC-5 medium containing 8 µg/ml of polybrene. The plates were returned to the incubator after adding an additional 1 ml of medium. Forty-eight hours after the second spin-infection, the fibroblasts were split at a ratio of 1:6 and maintained for 3 more days in MRC-5 medium before seeding for reprogramming on mouse embryonic feeder cells.

Preparation of mouse ES feeder cells

SNL 76/7-4 cells were grown to about 90% confluency, washed once with PBS and incubated in SNL growth medium with 10 µg/ml Mitomycin C (Sigma, Saint Louis, USA; Catalog number M4287) for 2 h at 37°C. The cells were washed three times in excess PBS, released with Trypsin-EDTA and frozen in aliquots and stored in liquid nitrogen.

Reprogramming of MRC-5 cells after transduction

In preparation for reprogramming, 6-well plates were coated with gelatin (0.1%, Millipore Corp., USA; Catalog number ES-006-B) overnight at 37°C. The next-day Mitomycin-treated mouse ES feeder cells (SNL 76/7-4) were seeded onto 6-well plates (500,000 cells/well). The following day, each well received 10,000 transduced MRC-5 fibroblasts in MRC-5 medium. The medium was changed the following day to human ES reprogramming medium (Knockout DMEM/F12 containing 20% Knockout serum replacer (KOSR), 100 µM non-essential amino acids, 100 µM 2-mercaptoethanol, 1 mM L-glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, and 10 ng/ml basic fibroblast growth factor (bFGF, Millipore, Massachusetts, USA). The medium was replaced every other day for about 10 days and then replaced daily for additional one to 2 weeks until putative iPSC-like colonies appeared and grew to a size that could be manually picked. The iPSC-like colonies were marked using an object marker (BioIndustrial Products, USA; Catalog number 14361) and picked using drawn out Pasteur pipets in a laminar floor hood under a stereomicroscope. Picked colonies were deposited on Mitomycin C-treated SNL feeder layer containing cells in either 24-well or 6-well plates. In later experiments we determined that we could directly transfer iPSC-like clones onto feeder-free Matrigel-coated (Beckton-Dickinson, USA) wells. The cells were gradually transitioned to a feeder-free defined medium (mTeSR1; STEMCELL Technologies, Vancouver, Canada), over a 4-day period by increasing its percentage by 25% each day (Neely et al., 2011). Individual clones were expanded and frozen down in aliquots using mFreSR (STEMCELL Technologies, Vancouver, Canada) as per the manufacturer’s recommended protocol.

Alkaline phosphatase

The iPSC clones were stained for the alkaline phosphatase pluripotency marker using either fixed or live cultures. For fixed cultures we used a kit from Sigma (Saint Louis, Missouri, USA; Catalog number 85L1-1KT), while for unfixed cultures we used the Alkaline Phosphatase Live Stain (Molecular Probes/Life Technologies, Grand Island, NY, USA; Catalog number A14353) as per the manufacturers’ recommended protocols.

Immunofluorescence assay for stem-cell markers

The putative iPSC clones were assessed for the expression of SSEA3, SSEA4, Tra-160, Tra-1-81 surface antigens and Oct4 and Nanog nuclear transcription factors using primary and fluorescent-labeled secondary antibodies as described previously (Neely et al., 2011).

Reverse-transcriptase-qPCR (RT-qPCR)

Expression of pluripotency marker genes (NANOG and DNMT3B), and RFs (OCT4, SOX2, KLF4 and cMYC), was determined using RT-qPCR of total mRNA isolated from individual IPSC clones using the primers listed in Table 3. The endogenous RF mRNA (RF_E) that originated from the cell was estimated using primers targeting either the 3′ or 5′ untranslated region. The total RF mRNA (RF_T) was quantitated using primers situated in the coding exon and measured both endogenous and vector-originated mRNAs. Some of the primers used in this study were originally published by Chan and coworkers (Chan et al., 2009) and subsequently validated by us in other studies (Neely et al., 2011). The reverse transcription was done with Superscript III kit (Invitrogen/Life Technologies, Grand Island, NY, USA; Catalog number: 11752250) using random hexamers according to the recommended protocol. The following cycling parameters were used for qPCR: Incubation at 95°C for 10 min, followed by 40 two-step cycles each consisting of 95°C for 15 s (denaturation) and 60°C for 60 s (annealing and extension). The qPCR was accomplished using the Power SYBR Green Master Mix (Invitrogen/Life Technologies, Grand Island, NY, USA; Catalog number: 4367659) in a 7900HT PCR System (Applied Biosystems).

Methylation-sensitive restriction enzyme qPCR (MSRE-qPCR)

Genomic DNA (60 ng), isolated using DNeasy Kits, was digested with 5 units of SmaI (methylation sensitive) or MscI (methylation insensitive) or incubated in the absence of restriction enzyme (uncut) in the manufacturer supplied buffer (New England Biolabs, Massachusetts, USA) in a 50 µl reaction volume at 37°C for 4 h. The reaction was terminated by inactivating the enzymes at 80°C for 20 min. An aliquot of the digest (5 µl) containing 6 ng of genomic DNA was then used in qPCR using primers (SK160 and SK161, Table 1) targeting the 5′ Moloney murine leukemia virus LTR and 5′ untranslated region (UTR) (Fig. 8A). The PCR was carried out using SsoAdvanced SYBR Green Supermix (Bio-Rad, Hercules, CA, USA; Catalog no. 1725261) in a Bio-Rad CFX96 thermocycler. The PCR cycling parameters consisted of a two-step amplification cycle consisting of a denaturation step at 98°C for 10 s and an annealing and extension step at 63.1°C for 30 s for a total of 40 cycles. A final melt-curve analysis was done as described above for vector titer determination. The vector copies were normalized to β-actin copy numbers present in the same samples as described earlier for vector titer determination.

Karyotyping

iPSC clones were karyotyped by Genetics Associates Inc., (Nashville, TN) or by the Cytogenetics Department at Saint Louis University Medical Center (Saint Louis University, Saint Louis, MO) using standard techniques.

Differentiation into ectodermal, mesodermal and endodermal elements through embryoid body (EB) formation (Ohnuki, Takahashi & Yamanaka, 2009)

Six-well plates containing iPSCs in log-phase of growth and showing large to medium sized colonies, were treated with Dispase (2 mg/ml in DMEM/F12, Invitrogen/Life Technologies, Grand Island, NY, USA) for 20 min at 37°C. The colonies were released by washing gently with the Dispase solution, and transferred to a 15 ml conical tube. The colonies were allowed to settle to the bottom of the tube at unit gravity, washed once by gently removing and replacing the supernatant with DMEM/F12, and then resuspended in EB formation medium (DMEM with 20% FBS or human ES reprogramming medium described in Materials and Methods but without bFGF). The colonies in EB formation medium were transferred to low-attachment T-25 flasks or 6-well plates (Corning, USA) and placed on an orbital shaker in 37°C humidified incubator. After 8 days, with alternate day medium change, the resultant EBs were transferred to 6-well plates coated with gelatin and allowed to attach and spread out. After additional 8 days, with regular medium change every other day, the cells were fixed using 4% paraformaldehyde in phosphate buffered saline, pH 7.2 and stained for ectodermal (βIII-tubulin), mesodermal (α-smooth muscle actin (α-SMA) or desmin) or endodermal (Sox17 or α-fetoprotein (α-SMA)) marker expression (Neely et al., 2011).

Statistical calculations

The standard deviation of the ratio of means of target gene (g) mRNA levels to control β-actin (c) mRNA levels was calculated as follows: μ_g/μ_c × √[(σ_g/μ_g)² + (σ_c/μ_c)²] where μ = mean and σ = standard deviation (Wikipedia, 2013).

Preparation and characterization of vector stocks

Vector stocks for each of the reprogramming vectors (and the mRFP1 vector) were prepared by transient transfection of HEK293T cells as described in Materials and Methods. The Yamanaka reprogramming vectors, however, do not express a marker gene that would enable easy determination of vector titer. Thus, most reprogramming experiments for generation of iPSCs use a ‘blind’ approach for transduction of target cells with an assumption of vector titer. We determined the titer of each vector stock preparation by isolation of genomic DNA of transduced HEK293T cells followed by qPCR using primers that targeted the packaging sequence in Moloney murine leukemia virus (Table 1). The PCR product size was 231 bp long and the optimal annealing and extension temperature was determined to be 63.1°C in a preliminary temperature gradient PCR experiment. For amplification of a control cellular gene, we chose human β-actin primers that gave a product size of 250 bp. The same annealing-extension temperature was used for β-actin amplification. Standards for qPCR were prepared by diluting a retroviral vector plasmid DNA in genomic DNA isolated from untransduced control cells. β-actin standards were generated by serial 10-fold dilution of genomic DNA. The results of vector titer determination are shown in Table 2.

Optimization of transduction of MRC-5 fibroblasts

MRC-5 fibroblasts were initially transduced with the mRFP1 encoding retroviral vector alone to determine optimal MOI and transduction parameters. We tested two different MOI (2.5 and 5.0), and transduction at unit gravity (1 × g) or spin-transduction at 1,200 × g for 2 h at room-temperature. The efficiency of transduction was estimated by fluorescence microscopy (Fig. 1). The majority of MRC-5 fibroblasts were transduced at both MOIs. The fluorescence intensity was higher in the spin-infection cultures than that observed at unit gravity, suggesting higher transduction efficiencies. This could not be categorically determined since the plate used for transduction at normal gravity (1 × g) seemed to have slightly lower light transmission characteristics than that used for transduction at 1,200 × g (as indicated by comparison of phase contrast and fluorescence images between the two plates, Fig. 1). However, parallel transductions carried out on primary human keratinocytes clearly revealed the higher efficiencies of transduction at 1,200 × g and with an MOI of 5 (Fig. S1). To ensure that the maximal numbers of cells were transduced, we chose an MOI of 5 with a spin-transduction protocol for reprogramming of MRC-5 fibroblasts. This also ensured that virtually all cells expressed the mRFP1 marker gene following transduction.

Reprogramming of MRC-5 fibroblasts with Yamanaka vectors and detection of putative IPSC-like colonies based on differential mRFP1 expression

We combined appropriate volumes of the supernatants of the four reprogramming vectors and that of the mRFP1 encoding vectors to give an MOI of 5 for each vector and then pelleted the virus by ultracentrifugation. The pellet was resuspended in MRC-5 growth medium and used for transduction of the human fibroblast cells by two rounds of spin-transduction. The efficiency of transduction was determined as follows: (1) An aliquot of the transduced cells was analyzed by flow cytometry which indicated a transduction efficiency of 97% (Fig. 2A). (2) Photomicrographs of phase-contrast and fluorescence microscopy were obtained (Fig. 2B) and the number of cells in each was counted using the cell-counter feature of NIH ImageJ software. Approximately 95% appeared to be transduced by this approach. (3) Genomic DNA was isolated from untransduced and vector-transduced MRC5 and subjected to qPCR for quantitation of vector and β-actin copy numbers. From this, the average number of vector copies per cell was estimated. The results are shown in Fig. 8B and indicate that each cell contained around 25 copies of vector. This number is consistent with the use of an MOI of 5 for each of the five vectors in the transduction.

Following two rounds of transduction, and a brief period of expansion, the MRC-5 fibroblasts were plated on mouse ES feeder cells (SNL 76/7-4) (10,000 MRC-5 cells/well of 6-well plate), and the plates were observed on a regular basis in an inverted fluorescence microscope. The mRFP1 expressing MRC-5 cells exhibited a rapid expansion in culture as monitored by fluorescence microscopy. In the first two weeks after seeding on mouse feeder cells the colonies exhibited indistinct borders (Fig. 3). Between weeks 2 and 4, colonies with well-demarcated borders also appeared. Fluorescence microscopy revealed that the colonies with indistinct borders still expressed mRFP1, while the colonies with distinct borders were ‘dark’, i.e., did not express the marker gene (Fig. 4). Some colonies showed invasion by occasional mRFP1-positive fibroblast-like spindle shaped cells at their edges (Fig. 4). Phase-contrast microscopy revealed that the cells in mRFP1-negative colonies exhibited scant cytoplasm and had nuclei with prominent nucleoli that are characteristic of iPSCs (Fig. 4 and phase contrast image at 10× magnification on the last row of Fig. 4).

We identified 24 colonies with iPSC-like morphology that were negative for mRFP1 expression. This provided a calculated efficiency of iPSC generation of 0.04% ((24 ÷ 60,000 seeded cells) × 100). Twenty-one of these were chosen for further cloning. Fourteen colonies survived picking and could be adapted to feeder-free conditions on Matrigel. The subcloned iPSCs continued to exhibit silencing of mRFP1 (Fig. 5). Six clones were subjected to a battery of tests to determine if they exhibited iPSC characteristics (Materials and Methods).

All clones expressed pluripotency markers alkaline phosphatase, SSEA3, SSEA4, Nanog, Oct4, and Tra-1-60 (Fig. 6A and Figs. S2 through S6).

Retroviral expression of RFs is shut off following successful reprogramming

To determine the relative levels of RF_E (endogenous) and RF_T (endogenous and vector-derived) mRNA expression, total RNA from iPSCs and control cells, were reverse transcribed and amplified by qPCR. The mRNA levels were normalized to β-actin levels. The normalized expression patterns are shown in Fig. S7. Untransduced control MRC-5 fibroblasts expressed low levels of OCT4 and SOX2 but readily detectable levels of KLF4 and cMYC. Transduced MRC-5 cells (MRC-5 5V), in contrast, exhibited high levels of RF_T in comparison to RF_E mRNA levels. All four RFs were raised in putative iPSCs. Counterintuitively, RF_Es sometimes exceeded the RF_Ts for some RFs. To therefore control for possible differing efficiencies of amplification or reverse-transcription of RF_T vs RF_E mRNAs, we determined RF_T to RF_E ratio for all samples. We then normalized these ratios for each RF to that of hES cells in which the entire contribution to the RF_T mRNA must originate only from endogenous sources. These normalized data are shown in Fig. 7A and indicate that the putative iPSC clones exhibited an average RF_T/RF_E ratio of the four RFs comparable to that in hES cells (with less than 2-fold difference). In contrast, vector-transduced MRC-5 cells, prior to reprogramming, exhibited 21- to 9,017-fold higher RF_T/RF_E ratios than hES or iPSCs. The vector-derived RF expression was then significantly decreased as the endogenous cellular transcription of the corresponding factors was increased. We also determined the expression of other genes characteristic of iPSCs (NANOG, DNMT3B). These were expressed at high levels in hES and the putative iPSC clones but not in untransduced MRC-5 or vector-transduced MRC-5 prior to reprogramming (MRV-5 5V) (Fig. 7B). These results indicate that the fibroblasts were fully reprogrammed.

Methylation status of 5′ LTR of Moloney vector in transduced MRC5 cells and derived iPSC clones correlates with gene expression studies

Silencing of expression from Moloney LTR promoter has been associated with methylation at CpG islands. To determine the methylation status of the viral LTR promoter, we designed an MSRE-qPCR assay that spans SmaI (methylation sensitive) and MscI (methylation insensitive) restriction sites in the 5′ LTR (Fig. 8A). The sense primer was located in the U3 region of LTR while the antisense primer was located in the 5′ UTR downstream of the primer binding site (PBS). This ensured that only the 5′ LTR was amplified in the qPCR assay. Genomic DNA from each iPSC clone was isolated and then digested with SmaI or MscI or mock-digested in the appropriate buffer. An aliquot of the digest was subjected to qPCR as described in Materials and Methods. For generating methylation controls, HindIII-linearized vector plasmid (4 µg) was methylated in vitro using CpG methyltransferase (M.Sss1; 4 units/µg of DNA, New England Biolabs, Massachusetts, USA) in the supplied buffer containing S-adenosylmethionine for 2 h at 37°C. The methylation reaction was terminated by heat inactivation at 65°C for 20 min. Genomic DNA from untransduced MRC5 cells was spiked with an aliquot of unmethylated or methylated vector plasmid and used as positive and negative controls for digestion and qPCR with SmaI or MscI. Genomic DNA from MRC5 fibroblasts transduced with all four reprogramming vectors as well as mRFP1 encoding vector was also digested with SmaI or MscI or undigested and then subjected to qPCR as for the iPSC clones. Each of the samples were also tested by qPCR for human β-actin as described in Materials and Methods.

The results are shown in Fig. 8B and indicate that there were similar numbers of vector copies per cell for the parent transduced MRC-5 cells and the derived iPSC clones (22 to 25 copies/cell). In 5 of the 6 clones the number of vector copies per cell was identical between uncut and SmaI digested genomic DNA samples. The complete resistance to digestion by SmaI in these clones was consistent with methylation at the SmaI site. In clone 17, the genomic DNA was only partially resistant to SmaI. In contrast, in vector-transduced parent MRC-5 fibroblasts, the SmaI site was exquisitely sensitive to digestion, indicating that prior to reprogramming the site was not methylated. The in vitro methylated (MPC) and unmethylated vector plasmid (UPC) showed resistance and susceptibility to SmaI, respectively, as anticipated. In all cases, PCR amplification was significantly thwarted by predigestion with the methylation insensitive MscI in the same buffer as SmaI, indicating that the genomic DNA did not contain inhibitors to restriction enzyme digestion. These results corroborate the RT-qPCR results described above showing complete abrogation of vector derived gene expression in the tested iPSC clones. The results also explain the silencing of mRFP1 expression during reprogramming.

Further characterization of the clones (Figs. S2–S6) revealed that with the exception of clone #16 (that showed trisomy of chromosome number 12, Fig. S4B), all other clones exhibited a normal karyotype (Fig. 6B and Figs. S2–S5). EBs derived from these clones (Fig. 9) generated cells that expressed markers of ectoderm, endoderm, and mesoderm (Fig. 6C and Figs. S2–S6C) supporting their pluripotency.