RNA-induction: A new method for iPS cell production

Author: Chris Kamel, 10/14/10

The reprogramming of differentiated adult cells into induced pluripotent stem (iPS) cells is accomplished by the expression of a small number of key genes. This is typically done by introducing DNA either by transfection or with viral vectors. Current methods, unfortunately, are not very efficient and run the risk of mutagenesis as a result of the inserted DNA integrating into the genome. While iPS cells are important tools for drug development and disease modeling, the aforementioned roadblocks limit clinical usefulness. To that end, recent work from Derrick Rossi’s lab at Harvard, published in Cell Stem Cell, uses a virus-free messenger RNA (mRNA) system to efficiently create iPS cells without the risk of genome integration.

Rossi’s group generated synthetic mRNAs expressing each of the four required genes, modified to avoid the cell’s anti-viral response. (Many viruses are RNA-based, so the cell has evolved defenses to deal with foreign RNA.) When introduced to the cell, these RNAs are stable enough to produce the necessary proteins, but degrade quickly so they pose no long-term problems. Initial experiments showed this system could be used to simultaneously express GFP and another marker protein, and that these proteins could be localized to different cellular compartments. The team was also able to successfully express proteins at sufficient levels to induce differentiation of mouse embryonic cells, demonstrating that the modified mRNA system can effectively direct cell fate.

Having established that these mRNAs were able to transiently express multiple proteins, the Harvard team tried using them to generate iPS cells, demonstrating transient expression of each of the four desired genes, conversion of cells to an ES-cell like morphology, and increased transcription of several pluripotency markers to levels comparable to embryonic stem cells. Transcriptional profiles showed that the mRNA-derived iPS cells were more similar to ES cells than those produced by viral methods. This is particularly interesting in light of recent research that showed that iPS cells retain some “memory” of their parental lines. If these iPS cells are closer to ES cells, perhaps they don’t suffer the same restricted differentiation potential, yielding a more robust pluripotent cell. Finally, each of the derived cell lines was able to differentiate into different cell lineages.

In addition to generating more ES-like cells than viral protocols, the RNA-induction method was also more efficient. Induced pluripotent cells emerged in a shorter amount of time (13 versus 24 days) and were obtained at a higher rate — almost 40 times more iPS cells were obtained by this method.

RNA-induced pluripotent stem cells are a way to obtain pluripotent cells from differentiated tissue at a higher efficiency and with less risk than existing protocols. Importantly, this method of using modified RNA for protein expression isn’t limited to iPS production, but can potentially be used to safely deliver proteins in other contexts as well, for example in cystic fibrosis. Patients with cystic fibrosis have mutations in the CFTR gene, and lack of this protein prevents proper ion transport across cell membranes resulting in the disease. Though it may be some time before we can achieve widespread delivery of these synthetic mRNAs in a patient, the methods described here open the possibility of replacing this protein as an avenue for potential future therapy.

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Chris Kamel

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2 Responses

  1. Paul Krzyzanowski says:

    What is exciting about the Rossi paper is the way they modify normal protein coding RNAs to help the molecules evade destruction in normal cells.You leave off on the idea of delivering CFTR to a Cystic Fibrosis patient with these RNAs, which is a great example of an application for RNAs (i.e. supplementing a mutated or deleted gene). However, this kind of technology will still restricted to the lab for the near future as delivery of RNAs in a clinical context is something that still challenges the RNA biotech industry.A few technologies that can help to target and deliver RNA payloads (i.e. siRNAs, shRNAs, mRNAs) to a particular location, such as packaging in viral envelopes that infect the target cells or generating RNAs attached to protein or RNA based antibodies that recognize a cell specific receptor. Except for a few examples where RNAs can be delivered directly, such as the respiratory system, specific and effective delivery technologies are the missing link in making of effective RNA drugs.

  2. Chris Kamel says:

    Hi Paul,Good points. I alluded to them in the OP, thanks for expanding – and I hope I didn’t imply this tech was going to the clinic right away.The use of viruses or protein attachments (eg. HIV-Tat) immediately sprung to mind when considering possible delivery, though both may be an odd choice for this particular system. The paper is quite explicit that a significant benefit of the modified mRNAs is avoiding the use of viruses and inefficiencies of proteins for transduction. Viral envelopes may avoid integration issues but still raise issues of immune response. You’re right that delivery is a bottleneck, though I doubt it will be long before we start seeing papers optimizing delivery of these modified mRNAs.

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