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Induced Pluripotent Stem Cells (iPS)

iPS Cell Reprogramming Using Modified mRNA and B18R

iPS Cells
Reprogramming
Benefits of RNA Delivery for iPS Reprogramming
B18R Introduction
iPS Reprogramming using mRNA and B18R Notes
Why Use B18R?

Validated mRNA/B18R Protocol


iPS Cells

Pluripotent stem cell lines can be obtained through the reprogramming of somatic cells from different tissues and species by ectopic expression of defined factors. Induced Pluripotent Stem cells (iPSCs) are suitable for various purposes, including:

  • disease modeling
  • autologous cell therapy
  • drug or toxicity screening
  • basic research

Recent methodological improvements are increasing the ease and efficiency of reprogramming, and reducing the genomic modifications required to complete the process.  Depending on the downstream applications, certain reprogramming technologies have advantages over others.


Reprogramming

Self-renewal and pluripotency are defining properties of embryonic stem cells (ESCs). They refer, respectively, to the ability to proliferate indefinitely without commitment in vitro and to the capacity to differentiate into cell lineages belonging to the three embryonic germ layers.
Reprogramming entails the trans-expression in a somatic cell of a set of core pluripotency-related transcription factors (OSKM). In most cases:

When successful, tightly compacted colonies called embryoid bodies appear on the culture dish. These clusters resemble ESCs morphologically, molecularly and phenotypically. iPSC’s represent a widely available, non-controversial and practically infinite source of pluripotent cells. Unlike human ESCs, their usage is not restricted, so most laboratories can now develop research programs using human iPS-cell lines.

Reprogramming requires delivery of certain factors (RNA) into a somatic cell and their adequate expression under defined culture conditions for a period of time. Depending on the donor cell type, reprogramming is achieved with different efficiencies and kinetics. For example, 8–12 days are required to reprogram mouse embryonic fibroblasts (MEFs) using retroviruses. The same process takes 20–25 days for human foreskin fibroblasts (HFFs). So far, fibroblasts remain the most popular donor cell type, and were used in more than 80% of all reprogramming experiments published.


Benefits of RNA Delivery for iPS Reprogramming

  • Completely eliminate plasmid or viral vectors.
    • Warren et al. developed a system that achieves the efficient conversion of different human somatic donor cells into iPSCs using direct delivery of synthetic mRNAs.
  • Much higher efficiency than achieved with other non-integrative systems.

B18R Introduction

B18R is a protein from the vaccinia virus first clone sin 1990 by Ueda et al for its ability to block viral potency by acting as a decoy receptor for Type I Interferons (IFNa, IFNb, IFNe,k,t,d,z, w,v) . B18R was later found by the Rossi lab to enable increased cell viability during RNA transfection protocols designed to convert human somatic donor cells into iPSCs via direct delivery of synthetic mRNAs.


iPS Reprogramming Using mRNA and B18R Notes

This protocol requires modification of in vitro transcribed RNAs in order for them to escape the endogenous anti-viral cell defense response to ssRNA.

  • RNA modification: Phosphatase treatment and incorporation of modified ribonucleoside bases substituting 5 methylcytidine for cytidine and pseudouridine for uridine.
  • Tissue Culture: Addition of a recombinant B18R protein in the medium.
    • Allows for high dose-dependent levels of protein expression with high cell viability.
    • Can allow reprogramming by serial transfection of different donor populations using a cationic vehicle (continuous use of B18R).

Provides an extremely appealing method due to its simplicity and efficiency of modified RNA.


Why use B18R?

B18R increases cell viability by suppressing IFN induced apoptosis from RNA transfection.

B18R, when combined with modified RNA (mRNA):

  • Can enable highly efficient reprogramming of somatic cells to pluripotency.
  • Allow for re-directed differentiation toward a desired lineage.
  • Removes the risk of genomic integration and insertional mutagenesis inherent to DNA-bases methodologies.
  • Eliminates the need for virus-based approaches.