An Improved Method to Generate Virus-Free Human iPSCs

Induced pluripotent stem cells (iPSCs) are offering new opportunities in both research and clinical applications. Researchers are using these cells for drug and toxicity screening, as well as for studying differentiation pathways and modeling diseases. The research has also led to insights that are helping to create patient-specific cell therapies and a more personalized approach to medicine. Though researchers are using iPSCs for a wide range of applications, better methods are needed to generate these cells; a ready source of iPSCs is critical for effective research and eventual therapeutic use.

It was discovered in 2006 that human iPSCs could be generated by inducing expression of the four reprogramming factors (OCT-4, SOX-2, KLF-4 and c-MYC).¹ Since then, many different reprogramming technologies have emerged to generate iPSCs, each possessing its own advantages and disadvantages.² First-generation technologies, based on retroviral and lentiviral systems, allowed for highly efficient reprogramming events but lacked the necessary control over host genome integrations. Cre-excisable lentiviral systems offered a solution to genome integration but required lengthy subcloning procedures and screening to ensure excision of the reprogramming factors.

Second-generation technologies used nonintegrating episomal DNA plasmids, which were transgene-free but lacked the high reprogramming efficiencies of earlier retroviral and lentiviral techniques. Third-generation technologies used negative sense, nonintegrating RNA viruses, termed Sendai Viruses (SeV), which originated from highly transmissible respiratory tract infections in mice, hamsters, guinea pigs, rats and pigs. These RNA viruses produced integration-free iPSCs, offered high reprogramming efficiencies, and were easy to use, but residual Sendai Virus was difficult to clear from cells, resulting in the requirement for multiple rounds of clonal expansion and analysis. Figure 1 depicts this evolution of reprogramming technologies.

Figure 1 ‒ The evolution of reprogramming technologies has culminated in the development of synthetic RNA-mediated reprogramming (extreme right), representing the safest and most efficient method for iPS cell generation. (Illustration adapted from Bernal, J.A. J. Cardiovasc. Trans. Res. 2013, 6, 956–68.)

The Simplicon™ RNA Reprogramming Technology from EMD Millipore (Billerica, Mass.) is a new reprogramming system that uses a single synthetic, polycistronic self-replicating RNA replicon engineered to mimic cellular RNA to generate human iPSCs.³ The single RNA strand contains the four reprogramming factors—OCT-4, KLF-4, SOX-2 and GLIS1—and enables efficient reprogramming using a single transfection step without any viral intermediates or host genome integration. Once iPSCs are generated, the RNA can easily be selectively eliminated by removing the interferon-gamma (IFNg) inhibitor, B18R, from the cell culture medium. The result is transgene-free, replicon-free iPSCs.

Generation of human iPSCs

First, 4 × 10⁵ human foreskin fibroblasts (HFFs) were plated in each well of a six-well plate in low serum fibroblast medium and allowed to attach overnight. HFFs were pretreated with B18R growth factor for 2 hours at 37 °C and 5% CO₂. HFFs were then transfected with 1 μg of Simplicon VEE-OKS-iG and B18R RNA in 2.5 μL of Lipofectamine^® 2000 transfection reagent diluted with Opti-MEM™ medium (Life Technologies, Grand Island, NY) following the manufacturer’s protocol. The mixture of Simplicon RNA and transfection reagent was incubated at 37 °C and 5% CO₂ for 3 hours. Following transfection with RNA, medium was exchanged with 2 mL/well of ADMEM medium containing 10% fetal bovine serum (FBS), 1% GlutaMAX™ supplement (Life Technologies) and B18R protein (200 ng/mL).

Starting the day after transfection, cells were fed daily with ADMEM with 10% FBS, 1% GlutaMAX supplement, B18R protein and 0.5 μg/mL puromycin for a total of 10 days. From days 4‒5, 30‒60% cell death was observed, and puromycin-resistant cells started to grow back at days 7‒9 after puromycin selection.

At day 10, approximately 5 × 10⁴ to 1 × 10⁵ reprogrammed cells were replated on fresh EmbryoMax^® Primary Mouse Embryo Fibroblasts (EMD Millipore) in MEF-conditioned medium containing B18R protein (200 ng/mL) supplemented with small molecules contained in the Human iPS Reprogramming Boost Supplement II (EMD Millipore). Cell morphology was monitored daily, and small iPSC colonies started to appear around days 15‒16. At day 20, reprogrammed cells were transitioned to standard human embryonic stem cell medium without B18R protein, and colonies were selected based on colony morphology and expanded for future experiments. Figure 2 provides a timeline showing when each reprogramming step was performed.

Figure 2 ‒ Timeline showing points at which Simplicon Reprogramming steps were performed.

Results

Transfection efficiency

Figure 3 ‒ Fluorescent micrographs of HFFs transfected with a GFP RNA replicon control (a) or Simplicon Reprogramming RNA (b) and analyzed the following day to estimate transfection efficiency. The green signal in (b) reflects OCT-4 expression.

HFFs were transfected with either a control RNA replicon encoding green fluorescent protein (GFP) alone (Figure 3a) or with the Simplicon Reprogramming RNA (Figure 3b). The cells were analyzed the following day to determine transfection efficiency. A high percentage of cells in Figure 3a showed GFP signal; transfection of HFFs using Simplicon Reprogramming RNA resulted in roughly 5‒10% Oct-4 expression one day after transfection (green, Figure 3b).

Figure 4 ‒ Brightfield microscopy shows HFFs being reprogrammed over a 21-day time period using a single transfection of Simplicon Reprogramming RNA. After 10 days of puromycin selection and replating on fresh PMEF cells, colonies started to emerge at days 17‒21.

Reprogramming kinetics

To determine the kinetics of reprogramming, Simplicon transfected HFFs were analyzed at four timepoints by brightfield microscopy (Figure 4). Colonies of human iPSCs emerged between days 17 and 21 after a single transfection at day 1.

Characterization of human iPSCs

Simplicon reprogrammed fibroblasts were assayed for alkaline phosphatase activity,   a pluripotency marker, 28 days after transfection (Figure 5). Both HFF and BJ fibroblasts showed high expression levels of alkaline phosphatase activity, with the HFF cells showing the most intense signal.

Figure 5 ‒ Alkaline phosphatase staining of day 28 reprogrammed fibroblasts (HFF and BJ) generated using Simplicon RNA Reprogramming technology.

Lot-to-lot reproducibility

To compare the performance of different manufacturing lots of the synthetic Simplicon RNA strands, four different lots were utilized to transfect HFFs, using 10,000 cells per transfection. The number of hiPSC colonies that appeared 17‒21 days later was then counted. By calculating the reprogramming efficiency as the number of reprogrammed colonies divided by the number of original cells, it was determined that all the manufacturing lots displayed similar reprogramming efficiencies, ranging from 0.5% to 1%.

Conclusion

This article describes a synthetic, polycistronic RNA-based technology that enables efficient reprogramming of human somatic cells into induced pluripotent stem cells using a single transfection event. The Simplicon RNA Reprogramming technology does not rely on lentiviral, retroviral, or RNA-based viruses, but combines the efficiency of retroviral and lentiviral reprogramming technologies with the safety of nonviral-based reprogramming methods. The technology is also a step toward a more defined system for iPSC generation.

In conclusion, this RNA-based reprogramming approach has broad applicability for the efficient generation of hiPSCs. The technique can be used to aid disease cell-modeling studies, transdifferentiation research and eventual human cell therapy and regenerative medicine applications.

References

1. Yamanaka, S. and Takahashi, K. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell  2006, 126(4), 663‒76.
2. Zhu, S.; Li, W. et al. Reprogramming of human primary somatic cells by OCT4 and chemical compounds. Cell StemCell  2010, 7(6), 651‒5.
3. Yoshioka, N.; Gros, E. et al. Efficient generation of human iPSCs by a synthetic self-replicative RNA. Cell Stem Cell  2013, 13(2), 246‒54.

Vi Chu is R&D manager, Nick Asbrock is product manager, and Min Lu is R&D scientist, EMD Millipore, 28820 Single Oak Dr., Temecula, CA 92590, U.S.A.; tel.: 951-514-4267; e- mail: [email protected]; www.emdmillipore.com