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Highly efficient induction of primate iPS cells by combining RNA transfection and chemical compounds.

ABSTRACT: Induced pluripotent stem (iPS) cells hold great promise for regenerative medicine and the treatment of various diseases. Before proceeding to clinical trials, it is important to test the efficacy and safety of iPS cell-based treatments using experimental animals. The common marmoset is a new world monkey widely used in biomedical studies. However, efficient methods that could generate iPS cells from a variety of cells have not been established. Here, we report that marmoset cells are efficiently reprogrammed into iPS cells by combining RNA transfection and chemical compounds. Using this novel combination, we generate transgene integration-free marmoset iPS cells from a variety of cells that are difficult to reprogram using conventional RNA transfection method. Furthermore, we show this is similarly effective for human and cynomolgus monkey iPS cell generation. Thus, the addition of chemical compounds during RNA transfection greatly facilitates reprogramming and efficient generation of completely integration-free safe iPS cells in primates, particularly from difficult-to-reprogram cells.

SUBMITTER: Watanabe T

PROVIDER: S-EPMC6852476 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Highly efficient induction of primate iPS cells by combining RNA transfection and chemical compounds.

Watanabe Toshiaki T Yamazaki Shun S Yoneda Nao N Shinohara Haruka H Tomioka Ikuo I Higuchi Yuichiro Y Yagoto Mika M Ema Masatsugu M Suemizu Hiroshi H Kawai Kenji K Sasaki Erika E

Genes to cells : devoted to molecular & cellular mechanisms 20190617 7

Induced pluripotent stem (iPS) cells hold great promise for regenerative medicine and the treatment of various diseases. Before proceeding to clinical trials, it is important to test the efficacy and safety of iPS cell-based treatments using experimental animals. The common marmoset is a new world monkey widely used in biomedical studies. However, efficient methods that could generate iPS cells from a variety of cells have not been established. Here, we report that marmoset cells are efficiently ...[more]

PMID: 31099158

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Project description:BackgroundThe discovery and development of RNA interference has made a tremendous contribution to the biochemical and biomedical field. However, liposomal transfection protocols to deliver siRNAs to certain types of cells, eg, immune cells, are not viable due to exceedingly low transfection efficiency. While viral delivery and electroporation are two widely adopted approaches to transfect immune cells, they are associated with certain drawbacks such as complexity of preparation, biosafety issues, and high cytotoxicity. We believe amendments can be made to liposomal formulas and protocols to achieve a highly efficient knockdown of genes by liposome-loaded siRNAs.AimThe aim of this study was to use the apoptotic-mimic Ca-PS lipopolyplex to achieve highly efficient siRNA knockdown of genes in the hard-to-transfect macrophages with reduced cytotoxicity and more efficient cellular uptake.ResultsWe devised an anionic liposomal formula containing phosphatidylserine to mimic the apoptotic body, the Ca-PS lipopolyplex. Ca-PS lipopolyplex was proven to be capable of delivering and effecting efficient gene knockdown in multiple cell lines at lowered cytotoxicity. Among the two types of macrophages, namely Ana-1 and bone-marrow derived macrophages, Ca-PS lipopolyplex showed an improvement in knockdown efficiency, as high as 157%, over Lipo2000. Further investigations revealed that Ca-PS promotes increased cellular uptake, lysosomal escape and localization of siRNAs to the perinuclear regions in macrophages. Lastly, transfection by Ca-PS lipopolyplex did not induce spontaneous polarization of macrophages.ConclusionThe apoptotic body-mimic Ca-PS lipopolyplex is a stable, non-cytotoxic liposomal delivery system for siRNAs featuring vastly improved potency for macrophages and lowered cytotoxicity. It is speculated that Ca-PS lipopolyplex can be applied to other immune cells such as T cells and DC cells, but further research efforts are required to explore its promising potentials.

Project description:Adeno-associated virus (AAV) has emerged as the preferred vector for targeting gene expression to the retina. Subretinally injected AAV can efficiently transduce retinal pigment epithelium and photoreceptors in primate retina. Inner and middle primate retina can be transduced by intravitreally delivered AAV, but with low efficiency. This is due to dilution of vector, potential neutralization of capsid because it is not confined to the immune-privileged retinal compartment, and the presence of the inner limiting membrane (ILM), a barrier separating the vitreous from the neural retina. We here describe a novel "subILM" injection method that addresses all three issues. Specifically, vector is placed in a surgically induced, hydrodissected space between the ILM and neural retina. In an initial experiment, we injected viscoelastic (Healon(®)), a substance we confirmed was biocompatible with AAV, to create a subILM bleb and subsequently injected AAV2-GFP into the bleb after irrigation with basic salt solution. For later experiments, we used a Healon-AAV mixture to place single, subILM injections. In all cases, subILM delivery of AAV was well tolerated-no inflammation or gross structural changes were observed by ophthalmological examination or optical coherence tomography. In-life fluorescence imaging revealed profound transgene expression within the area of the subILM injection bleb that persisted for the study duration. Uniform and extensive transduction of retinal ganglion cells (RGCs) was achieved in the areas beneath the subILM bleb. Transduction of Müller glia, ON bipolar cells, and photoreceptors was also observed. Robust central labeling from green fluorescent protein-expressing RGCs confirmed their continued survival, and was observed in the lateral geniculate nucleus, the superior colliculus, and the pretectum. Our results confirm that the ILM is a major barrier to transduction by AAV in primate retina and that, when it is circumvented, the efficiency and depth to which AAV2 promotes transduction of multiple retinal cell classes are greatly enhanced.

Dataset Information

Highly efficient induction of primate iPS cells by combining RNA transfection and chemical compounds.

Publications

Highly efficient induction of primate iPS cells by combining RNA transfection and chemical compounds.

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