Life Science


by Vi Chu, Nick Asbrock and Min Lu


An Improved Method to Generate Virus-Free Human iPSCs


I


nduced pluripotent stem cells (iPSCs) are offering new opportuni- ties 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 induc- ing expression of the four reprogramming factors (OCT-4, SOX-2, KLF-4 and c-MYC).1


have emerged to generate iPSCs, each possessing its own advantages and disadvantages.2


Since then, many different reprogramming technologies 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 respira- tory 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.


The Simplicon RNA Reprogramming Technology from EMD Millipore (Billerica, Mass.) is a new reprogramming system that uses a single syn- thetic, 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 with- out 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


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 gen- eration. (Illustration adapted from Bernal, J.A. J. Cardiovasc. Trans. Res. 2013, 6, 956–68.)


AMERICAN LABORATORY • 42 • MARCH 2015 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, N.Y.) 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 morphol- ogy 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


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