Reprogramming of Human Fibroblasts using Non-Integrating Self-Replicating RNA Vectors
Reprogramming of Peripheral Blood Mononuclear Cells (PBMCs) using STEMCCA Lentiviral Vectors
Characterization and Picking of Reprogrammed iPSC Colonies
Frequently Asked Questions
A ready source of induced pluripotent stem cells (iPSCs) is critical to the effective study of differentiation pathways and the investigation of the therapeutic potential of iPS cells. Since the discovery that human iPSCs could be generated by inducing expression of the four reprogramming factors (OCT-4, SOX-2, KLF-4 and c-MYC)1 many different reprogramming technologies have emerged to generate iPSCs, each possessing their own advantages and disadvantages2.
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 non-integrating 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, non-integrating 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, produced 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.
Next generation reprogramming system that use synthetic self-replicating RNA engineered to mimic cellular RNA have been used to generate human iPS cells3. The single RNA strand contains the four reprogramming factors and enables extremely 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.
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Figure 1. Evolution of Reprogramming Method.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: Simplicon™ RNA Reprogramming Technology combines the efficiency of retroviral and lentiviral reprogramming technologies with the safety of non-viral based reprogramming methods.
Figure 2. Simplicon RNA Human HFF Reprogramming Timeline.Using a single transfection of self-replicating RNA can produce high numbers of integration-free iPSCs following a 3-4 week protocol.
Figure 3. Time course of human iPSC colonies generation using Human Simplicon RNA Reprogramming Kit.The transfected HFFs were replated onto inactive MEFs at Day 10, Colonies start to emerge from Day 15-16 and are more obvious around day 17-20 (A, B). Colonies are ready to be picked at Day 26.
Recent findings indicate that fully reprogrammed human iPS cells possess the following characteristics: (1) they downregulate expression of fibroblast marker, CD13 (2) they upregulate expression of pluripotent markers, SSEA-4 and TRA-1-60, (3) they silence the viral transgenes while (4) reactivating endogenous expression of Nanog and (5) they assume a HoeschstDim phenotype. These characteristics have enabled reliable identification and colony picking of fully reprogrammed human iPS cells from a mixed population of partially reprogrammed cells.
Millipore’s Human iPS Selection Kit (SCR502) is a quick, easy and non-invasive method to monitor the pluripotent state of fully reprogrammed human iPS cells using immunocytochemistry (ICC). The Human iPS Selection Kit allows live cell imaging and identification of fully reprogrammed human iPS cells from a heterogeneous population of reprogramming intermediates and enables the selection of human iPS cells that can be further passaged and expanded for downstream applications.
Figure 4.Fully reprogrammed human iPS cells express human pluripotent markers, TRA-1-60 FITC (D, E, G, H, green) and SSEA-4 PE (C, F, G, H, red) while downregulating the fibroblast marker, CD13 PE (data not shown). Cells were stained with cell permeable Hoechst nuclear dye (B, C, D, H, blue). Fully reprogrammed human iPS cells exhibit Hoechst dim phenotype (see colony center in B, C, D, H) while non-iPS and differentiated cells exhibit a Hoechst bright phenotype (see the periphery of the colony in B, C, D, H, which is surrounded by fibroblast cells and are Hoechst bright). Human iPS colonies at passage 5 were used for live staining.