Efficient generation of transgene-free human induced pluripotent stem cells (iPSCs) by temperature-sensitive Sendai virus vectors

Abstract

After the first report of induced pluripotent stem cells (iPSCs), considerable efforts have been made to develop more efficient methods for generating iPSCs without foreign gene insertions. Here we show that Sendai virus vector, an RNA virus vector that carries no risk of integrating into the host genome, is a practical solution for the efficient generation of safer iPSCs. We improved the Sendai virus vectors by introducing temperature-sensitive mutations so that the vectors could be easily removed at nonpermissive temperatures. Using these vectors enabled the efficient production of viral/factor-free iPSCs from both human fibroblasts and CD34(+) cord blood cells. Temperature-shift treatment was more effective in eliminating remaining viral vector-related genes. The resulting iPSCs expressed human embryonic stem cell markers and exhibited pluripotency. We suggest that generation of transgene-free iPSCs from cord blood cells should be an important step in providing allogeneic iPSC-derived therapy in the future.

Fig. 1.

Generation of TS SeV vectors and inactivation after temperature-shift treatment. (A) Point mutations were introduced into the polymerase-related genes P (P2: 433, 434, and 437) and/or L (942, 1361, and 1558), as indicated in the schematic structure of the ΔF/SeV vector. Open angles indicate conventional mutations in the previous TS vector; closed angles, newly introduced mutations. (B) Confluent LLC-MK2 cells were transduced with each SeV vector carrying GFP at an MOI of 5 and cultured at the indicated temperatures (32, 35, 37, 38, and 39 °C). Green fluorescence was compared at 3 d after infection. (C) To confirm the irreversible inactivation of gene expression by temperature-shift treatment, infected cells were cultured at 37 °C for 10 d and then split into two groups, one group cultured at 37 °C and the other cultured at 39 °C for 28 d, with cells passaged every 7 d. Similarly, cells infected with a TS vector treated at a nonpermissive temperature of 39 °C for 7 d were also cultured for a further 28 d at 37 °C, with cells passaged every 7 d, to evaluate GFP expression.

Fig. 2.

(A) Strategy used to obtain vector/factor-free iPSCs with an SeV vector mixture. Human fibroblasts were infected with the SeV vector mixture containing four factors. During reprogramming, polymerases may be supplied to the TS vector (which has c-MYC at the HNL position) by OCT3/4-, SOX2-, and KLF4-carrying conventional SeV vectors. Then the SeV vector may disappear when there is TS vector alone at nonpermissive temperature. (B) Procedure for reprogramming using SeV vectors. (C) qRT-PCR of existing SeV genomes in induced cells. RNA was extracted from cells between 3 d and 2 mo after infection, and the amount of SeV genome was analyzed by qRT-PCR. iPSC colonies were passaged every 7 d after day 35 (arrow), and whole colonies on the culture dish were analyzed. (D) Typical staining of iPSC colonies with anti-SeV antibodies. (Left) At passage (P) 1, many colonies were positive for SeV. (Middle) After several passages (P4), many colonies were partially positive. (Right) Colonies were found to be negative for SeV at P10. (Scale bar: 200 μm.)

Fig. 3.

(A) Ratio of SeV-positive colonies. P, passage number. (Left) Randomly chosen colonies were expanded independently, and the existence of SeV vectors was evaluated by qRT-PCR at P4 and P10. The number of positive and negative colonies was counted and is expressed for each as a ratio of all colonies chosen (n = 12 per each TS vector). (Right) Temperature shift to a nonpermissive temperature of 38 °C effectively removed SeV vectors from the iPSC colonies generated using TS7 vectors. The culture dishes at P4 were split and transferred to culture at 37 or 38 °C for the number of days indicated. The ratio was calculated as left panel. (B) SeV proteins were not detected in iPSC colonies by Western blot analysis with anti-SeV antibodies. Clone B1, positive control for iPSCs in which the SeV persisted (17); control, LLC-MK2 cells transfected with plasmids encoding SeV vectors. 5.5-3 and 5.5-5 were generated with MYC/TS7; 13-1, 13-5, and 13-6 were generated with MYC/TS13; and 15-3, 15-4, 15-5, and 15-6 were generated with MYC/TS15. (C) qRT-PCR of viral genome in SeV-generated iPSCs (viral genome/actin). BJ, parental fibroblasts; day 3 and day 7, BJ cells infected with SeV vectors after 3 and 7 d; HNL1 and HNLs, iPSCs established using conventional SeV (17). P8, pooled colonies at passage 8. Values <0.001 were backgrounds under the calculation curve as detected in parental cells. (D) Copy numbers of OCT3/4, SOX2, KLF4, and c-MYC in parental cells [BJ and human dermal fibroblast (HDF) cells] and iPSCs generated by SeV (SeV-iPS; n = 11) or retrovirus (retro-iPS; n = 6), as determined by qRT-PCR of genomic DNA. The passage numbers of tested clones are listed in Table S3.

Fig. 4.

Our second strategy with temperature-shift treatment. (A) Schemes to remove viral genome. (B) qRT-PCR of the SeV genome and hESC markers before and after temperature-shift treatment. iPSCs generated from HDF cells using TS7 vectors were treated with a temperature shift to 38 °C for 5 d. The SeV genome was not detected after this temperature-shift treatment, whereas expression of NANOG was not affected. (C) Schemes for generation of virus vector-free iPSCs from CD34⁺ CB cells with SeV vectors. The ESC-like colonies 4, 15, and 17 that emerged were subcloned after heat treatment for 3 d at 38 °C. (D and E) The remaining SeV construct in SeV iPSC 4, SeV iPSC 15, and SeV iPSC 17 (D; passage 2, before heat treatment) and heat-treated subclone SeV iPSC 4A (from 4), SeV iPSC 15A (from 15), and SeV iPSC 17A (from 17) (E; passage 4) were determined by immunostaining with anti-HN antibody (Left) and by qRT-PCR against endogenous NANOG transcript and SeV RNA construct (Right). Retrovirally generated iPSCs from CB cells (Rev) and CD34⁺ CB cells (CD34) were used as controls. The percentage of SeV-positive cells in the respective colony was determined using a two-value recognition function and is given below the qRT-PCR image.