A non-integrating, self-replicating RNA reprogramming vector for generating iPS cells
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Figure 1. Schematic of ReproRNA™-OKSGM, a Single‑Stranded RNA Replicon Vector
Figure 2. Schematic of Reprogramming Timeline with ReproRNA™-OKSGM
Somatic cells are transfected with ReproRNA™-OKSGM at day 0, and cultured in Advanced DMEM (AdvDMEM) with puromycin. After 6 days of puromycin selection post-transfection, cells are cultured in ReproTeSR™ for the remainder of the reprogramming induction phase until iPS cell colonies emerge. B18R recombinant protein is also added during the first 2 weeks after transfection to inhibit the interferon response and increase cell viability. Typically, by day 20, iPS cell colonies are large enough to be isolated and propagated in TeSR™ media. *TeSR™ = TeSR™ family media (mTeSR™1, TeSR™2, TeSR™-E8™).
Figure 3. ReproRNA™-OKSGM Vector Efficiently Reprograms Fibroblasts
Dermal fibroblasts were transfected with the ReproRNA™-OKSGM vector and reprogrammed under feeder-dependent (standard KOSR-containing hES cell medium on irradiated mouse embryonic fibroblasts (iMEFs)) or feeder-independent conditions (ReproTeSR™ on Corning® Matrigel®). Fibroblasts (passage 4) were reprogrammed with average efficiencies of 0.10 ± 0.03% (hES cell medium) and 0.20 ± 0.01% (ReproTeSR™). Reprogramming efficiency of fibroblasts with ReproRNA™ and ReproTeSR™ is comparable to that reported with Sendai virus.¹ (n ≥ 6; Data shown are mean ± SEM).
Figure 4. Feeder-Free Reprogramming with ReproRNA™-OKSGM Vector and ReproTeSR™ Generates iPS Cell Colonies with Superior Colony Morphology
Representative images of iPS cell colonies were generated using ReproRNA™‑OKSGM and cultured in (A) standard hES cell medium on irradiated mouse embryonic fibroblasts (iMEFs) or (B) ReproTeSR™ on Corning® Matrigel®. iPS cell colonies derived using ReproTeSR™ exhibit more defined borders, compact morphology, and reduced differentiation as compared to the ES cell medium.
Figure 5. Human iPS Cells Generated with ReproRNA™-OKSGM Express Undifferentiated Cell Markers
Human iPS cells generated with ReproRNA™-OKSGM display high expression of undifferentiated cell markers (OCT4 and TRA-1-60) as shown by flow cytometry analysis after 12 passages in mTeSR™1. (Filled histogram = sample, hollow histogram = secondary antibody only).
Figure 6. iPS Cells Derived Using ReproRNA™-OKSGM Display a Normal Karyotype
Karyogram of iPS cells derived with ReproRNA™-OKSGM and cultured in mTeSR™1 for 8 passages shows that a normal karyotype is retained.
Figure 7. ReproRNA™-OKSGM Derived iPS Cells Have the Capacity to Differentiate to Cells of the Three Germ Layers
Human iPS cells derived with ReproRNA™-OKSGM and maintained in mTeSR™1 for 7 passages were differentiated into cells of the three germ layers. Endoderm specification was achieved using the STEMdiff™ Definitive Endoderm Kit and flow cytometry analysis shows a high percentage of cells (98.7%) positive for endoderm markers (CXCR4+SOX17+). Mesoderm induction was achieved with STEMdiff™ Mesoderm Induction Medium as shown by the high percentage of cells (98.6%) expressing Brachyury (T). Ectoderm specification was demonstrated using STEMdiff™ Neural Induction Medium. CNS-enriched NPC cultures expressing PAX6 (green) and stained with DAPI (blue) are shown.
Protocols and Documentation
Find supporting information and directions for use in the Product Information Sheet or explore additional protocols below.
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