Virus-free induction of pluripotency and subsequent excision of reprogramming factors

Abstract

Reprogramming of somatic cells to pluripotency, thereby creating induced pluripotent stem (iPS) cells, promises to transform regenerative medicine. Most instances of direct reprogramming have been achieved by forced expression of defined factors using multiple viral vectors. However, such iPS cells contain a large number of viral vector integrations, any one of which could cause unpredictable genetic dysfunction. Whereas c-Myc is dispensable for reprogramming, complete elimination of the other exogenous factors is also desired because ectopic expression of either Oct4 (also known as Pou5f1) or Klf4 can induce dysplasia. Two transient transfection-reprogramming methods have been published to address this issue. However, the efficiency of both approaches is extremely low, and neither has been applied successfully to human cells so far. Here we show that non-viral transfection of a single multiprotein expression vector, which comprises the coding sequences of c-Myc, Klf4, Oct4 and Sox2 linked with 2A peptides, can reprogram both mouse and human fibroblasts. Moreover, the transgene can be removed once reprogramming has been achieved. iPS cells produced with this non-viral vector show robust expression of pluripotency markers, indicating a reprogrammed state confirmed functionally by in vitro differentiation assays and formation of adult chimaeric mice. When the single-vector reprogramming system was combined with a piggyBac transposon, we succeeded in establishing reprogrammed human cell lines from embryonic fibroblasts with robust expression of pluripotency markers. This system minimizes genome modification in iPS cells and enables complete elimination of exogenous reprogramming factors, efficiently providing iPS cells that are applicable to regenerative medicine, drug screening and the establishment of disease models.

Figure 1. Efficient reactivation of pluripotent markers in iPS cells generated by a non-viral multiprotein expression vector

a. Quantitative PCR for total and endogenous c-Myc, Klf4, Oct4 and Sox2 expression. Data is shown as relative expression to an ES cell line, E14Tg2a (E14). Error bars indicate the s.d. generated from triplicates. b. Quantitative PCR for pluripotent markers. Two independent ES cell lines, E14Tg2a (E14) and CGR8, were analyzed together with iPS cell lines. Data is shown as relative expression to E14Tg2a, and represents one of two independent experiments.

Figure 2. Non-viral iPS cells with a single vctor integration site

a. Schematic diagram of restriction enzyme sites, AflII and KpnI in pCAG2LMKOSimO and two probes, CAG probe and Orange probe, used for Southern blotting (bars). Black arrows (invAluR2, inv3TaqF1) indicate position of primers used to detect tandem repeat integration in c. b. Southern blotting analysis for AflII (top two panels) and KpnI (bottom two panels) digested genome of imO1-8 and TNGimO1-5, using CAG probe (left panels) and Orange probe (right panels). c. Validation of the integration site and tandem repeat integration. A single integration site of imO7 (white circle in b), and a single integration site with tandem integration (same orientation) of imO1 and imO3 (black circles in b) was identified by inverse PCR (data not shown) and validated by genomic PCR. Asterisk; band from wild-type allele. Arrowhead; integration site-specific band. Detail of the integration site is shown in Supplementary Figure 5. Note different band size of tandem repeats is caused by vector degradation accompanied by random integration.

Figure 3. Reprogramming cassette excision and pluripotency of the non-viral iPS cells

a. Many of the undifferentiated colonies at 5 days post Cre-transfection differentiated by day 9 in the absence of an Fgf receptor inhibitor, PD173074 (bottom). b. Percentage of reprogramming cassette-free undifferentiated colonies in the absence (−PD) and presence (+PD) of PD173074. Numbers of monitored reprogramming cassette-excised colonies are indicated in parentheses. Experiments were performed in three cell lines, imO2, imO7 and TNGimO5. c. Undifferentiated cells in imO7 teratoma (left) and various tissues in imO7c8 teratoma (the other three panels). d. imO3Ec5 derived chimeric embryos (green) at 10.5 dpc (left panels). e. Transversal sections at 9.5 dpc (upper six panels) and genital ridge at 12.5 dpc (bottom panels). imO3Ec5 contributed to ectoderm (neural tissue; n with magnification), mesoderm (heart; h), endoderm (gut; g with magnification) and germ cells (red staining with anti-Oct4 antibody). f. An adult imO7c8 derived chimeric mouse.