Cell reprogramming and iPS Cells engineering with FlashRNA^®

The delivery of different factors at once is crucial for lineage conversion. FlashRNA^®, as a multiple RNA transfer tool, is perfectly suited to human induced pluripotent stem cells (iPSCs) engineering and cell reprogramming. It allows a gentle RNA transfer, which preserves cell viability and the original iPSCs phenotype, safeguarding intact differentiation capacities.

FlashRNA^® for direct reprogramming of human fibroblasts

Thanks to its high payload capacity, FlashRNA^® can package and deliver the 4 reprogramming factors OCT4, SOX2, KLF4 and c-MYC at once. Human fibroblasts engineered with FlashRNA^®(O/S/K/M) show a high expression level of the 4 factors at 24h post-mRNA transfer.

FlashRNA^® efficiently triggers differentiation of primary human mesenchymal stem cells

FlashRNA^® promotes a cell-fate shift in immature human mesenchymal stem cells (MSC-l) by delivering two transcription factors involved in osteoblastic differentiation

mRNA quantification of MSC differentiation factors

Highly immature primary human MSC-l were engineered with FlashRNA^® carrying RUNX2 mRNA or DLX5 mRNA (5 pg p24/cell). Control cells were non-engineered and BMP4-treated to differentiate MSC-l into functional osteoblasts. 58 hours after FlashRNA^® transduction, RT-qPCR analyzes show that the mRNA expression levels of the two factors significantly increased, as well as the osteoblastic marker integrin-bone sialo-protein (iBSP). Conversely, the osteoprogenitor master factor OSTERIX showed no change in expression, suggesting a commitment of MSC-l toward an early osteoblastic lineage stage.

transcriptomics analyses showing MSC differentiation

Transcriptomics analyses on MSC-l following transduction with FlashRNA^®-DLX5 or FlashRNA^®-RUNX2, with a non-integrative lentiviral vector expressing Luciferase reporter (IDLV-Luc) as a reference. Data show upregulation of osteoblastic markers, confirming the commitment of cell differentiation toward an early osteoblastic lineage.

Reference: Prel, A., et al Mol. Ther. Methods Clin. Dev. 2015

FlashRNA^®: a highly potent yet gentle gene transfer tool for human iPS cells

FlashRNA^® mediates high-efficiency RNA transfer into human iPS cells for either transient gene overexpression or gene editing, while maintaining excellent cell viability and phenotype integrity.

Human HY03 iPS cells retained a pluripotent morphology and expressed pluripotency markers after transduction with FlashRNA^®, as indicated by the expression of NANOG, OCT3/4, SOX2, and SSEA4.

FlashRNA^® engineers human iPSC without altering their differentiation ability as they can be successfully differentiated into definitive endoderm, with a high expression of specific markers such as CXCR4 (cytometry plot), FOXA2 and SOX17 (immunofluorescence).

Reference: Mianné J, et al BMC Biol. 2022.

FAQ

How many RNA can be delivered with FlashRNA^®?

We have been able to transfer 4 different genes so far, but this is not the limit.

Is FlashRNA^® capable of engineering difficult-to-transduce cells ?

We achieve excellent transduction rates for all cell types, including those difficult to transduce, such as primary cells and stem cells. For difficult-to-transduce cells or sensitive cells, we recommend using the Premium production grade to achieve the highest transduction efficiency.

What is the duration of RNA expression with FlashRNA^®?

The RNA expression with FlashRNA^® is transient, meaning it lasts for a short period. The duration of expression is related to the half-life of the protein being expressed. Typically, for a short half-life reporter, protein expression is detectable from 4 hours post-transduction and reaches a peak after around 24 hours, before decreasing. For a long half-life reporter, expression can be detected up to 7 days after transduction.

Are the cells modified by FlashRNA^® considered as GMO?

The FlashRNA^®-modified cells are not considered as GMO, as the GOI expression is transient. There is one exception: if FlashRNA^® is used to deliver a genome editing system, like CRISPR/Cas9, that induces permanent genome modifications in the target cell.

More FAQs