Reprogramming adult cells, breakthrough by harvard stem cell institute

Reprogramming adult cells, breakthrough by harvard stem cell institute

Scientists have discovered a new way of creating stem cells from skin that has a much lower risk of cancer - in a report in the journal Cell Stem Cell the researchers say this is such a huge leap forward in reprogramming human adult cells that Harvard Stem Cell Institute will start using their new method to make patient and disease-specific induced pluripotent stem cells (iPS cells) straight away. Pluripotent stem cells can turn into any kind of human cell.

Doug Melton, co-chair of Harvard's Department of Stem Cell and Regenerative Biology, said:

This work by Derrick Rossi and his colleagues solves one of the major challenges we face in trying to use a patient's own cells to treat their disease. I predict that this will immediately become the preferred method to make iPS cells from patients and, indeed, at the HSCI we are turning our entire iPS core to using this method.

Derrick Rossi and team at the Immune Disease Institute at Children's Hospital Boston utilized synthetic mRNA to reprogram fibroblasts (adult human skin cells) and converted them into cells that appear to be identical to human embryonic stem cells. Embryonic stem cells can turn into any kind of cell, they are the building blocks of all human organs and tissue.

The researchers then used other mRNA to program new cells, which they have called RNA-iPS (RiPS), and created muscle cells. These cells should be able to turn into any kind of cell.

mRNA should be safe to use in treating patients, says Rossi. mRNA carries genetic instructions but does not penetrate the DNA of the target cells, unlike the iPS cells currently being created globally.

In an interview, Rossi said:

Our findings address three major impediments to clinical translational use of iPS cells. The method: does not in any way breach genomic integrity as it does not necessitate integrating genes or viruses into the target cells' DNA; it is orders of magnitude more efficient at producing iPS cells than conventional iPS methods, which were notoriously inefficient; and it gives us a way to directly program and direct the fate (development) of the iPS cells towards clinically useful cell types.

It appears the scientists have solved all three major problems which have baffled researchers ever since Shinya Yamanaka, a Japanese scientist, used four genes in 2006 to convert fibroblasts into cells with all the properties of embryonic stem cells. Yamanaka called them iPS cells (induced pluripotent stem cells) - they could be induced to become any type of cell in the human body.

However, Yamanaka used a virus to get the genes into the target cells' genome, which created at least two major obstacles when trying to use iPS cells for disease therapy:

  • Cancer risk - by integrating viruses cancers might unintentionally be triggered
  • Not identical to embryonic stem cells - by placing the genes inside the genome, changes could occur which would alter the properties of the resulting iPS cells.
Scientists worldwide have ever since been attempting to seek out other ways of turning adult cells into iPS cells in order to create cell lines that carry the genes of diseased patients so that they can study the disease, as well as to treat patients by creating patient-specific cell lines for them. Now, they may have what they were looking for, the researchers write.

Rossi explains:

Most approaches for generating iPS cells involve some sort of integration into the genome, usually viral. So clearly the development of a technology that does not breach genomic integrity is very important. Gene therapy trials unfortunately taught us the danger in leaving viruses in the genome as some patients developed cancers that were driven by the integrated viruses. So when one thinks about strategies for regenerative medicine, you need to envisage utilizing cells whose genome has not been breached. We believe that that utilizing RNA to generate transplantable cells and tissues is a ideal solution because,to the best of our knowledge, RNA is completely non-integrative.

In other words:

  • Rossi and team have created mRNA, an artificial messenger that carries the instructions sets from the four genes Yamanaka used. The mRNA instructs the adult cells to reprogram, in the same way Yamanaka's did, but this time without upsetting the integrity of the genome of the adult cell.
  • Yamaka managed to do that too, but he was unable to avoid upsetting the integrity of the genome of the adult cell.
The resulting RiPS do not have viral transgenes, so they are more identical to human embryonic stem cells. When Rossi and team compared their RiPS cells to human embryonic stem cells, they were much more similar compared to the iPS cells that were generated with viruses.

So, how to you convert RiPS cells into cells scientists need to treat patients, such as beta cells that are destroyed in diabetes Type 1?

Rossi explains:

Up to this point it's been extremely difficult to direct cells to differentiate towards particularly fates, or cell types

At the moment, in order to get cells to develop in the way you want, scientists have relied on carefully controlling the environment where the cells are developing, tailoring the growth media as well as other factors so that the iPS cells turn into a specific type of cell.

Rossi said:

We thought to use mRNA encoding cell type specific factors in order to drive the fate of iPS cells to the desired cell fate. We are beginning to know more about what factors are need to create certain types of cells - a great example was the demonstration by Doug Melton's group that they could use just 3 specific factors to turn adult pancreatic exocrine cells into insulin-producing beta cells.

However, those experiments needed a gene-carrying virus to be placed inside the target cell, Rossi said, although the Melton group used chemicals instead of some of the viruses.

To be able to show that mRNA could be used to direct which way an iPS cell evolves, Rossi and team synthesized an mRNA with the instruction set for making muscle cells, and demonstrated that they could use this to effectively direct the RiPS to become muscle cells - and without undermining the integrity of these cells genomes.

Rossi added:

These results provide us with a new experimental paradigm that might safely be used in regenerative medicine.

The researchers say they have found a method that is much more efficient than any previous one for making iPS cells.

Rossi continued:

Up until now, iPS cell generation has been an extremely inefficient process. Our technique allows for iPS generation that's significantly more efficient than conventional approaches.

Rossi and team say their iPS conversion efficiencies range from 1% to 4% of starting cells, compared to 0.001% to 0.01% (which used to be the case), meaning that if only very few starting cells are used, iPS cells can still be generated. This could be a crucial advantage if only a few starting cells can be obtained from a patient.

Rossi and colleagues say they have also found a way to overcome the natural cellular immunity to the insertion of foreign RNA.

Rossi said:

I am sure were not the only lab to have the idea of using RNA for cellular reprogramming. The problem is that when you introduce RNA into a cell, the cell thinks it is being infected by an RNA virus and retaliates by producing a massive interferon response that effectively shuts down cellular function and can prompt the cell to altruistic suicide as it tries to stop the 'virus' from replicating. In order to use RNA for cellular reprogramming we clearly needed to overcome this problem. Our approach was to modify the RNA so that it no longer set off anti-viral responses when introduced into cells. The modified-mRNA enabled us to efficiently express proteins in cells for days and weeks without causing any adverse reaction in the cells. This in turn allowed us to reprogram cell to pluripotency, which is a process that requires several weeks of Yamanaka factor expression.

"Although we developed this technology for cellular reprogramming, it actually has utility far beyond that. Basically our technology provides a means of transiently expressing any protein in a cell without eliciting the cell's anti-viral response pathways. This could have potential therapeutic benefit in patients suffering from a protein deficiencies.

Doug Melton said:

It's wonderful to see that HSCI seed grant funds given to outstanding, innovative, and imaginative young scientists like Rossi that can so quickly and dramatically change a field.

Click here to listen to Derrick Rossi and Doug Melton describe this work.

Source: Harvard Stem Cell Institute

"Highly Efficient Reprogramming to Pluripotency and Directed Differentiation of Human Cells with Synthetic Modified mRNA"

Luigi Warren, Philip D. Manos, Tim Ahfeldt, Yuin-Han Loh, Hu Li, Frank Lau, Wataru Ebina, Pankaj K. Mandal, Zachary D. Smith, Alexander Meissner, George Q. Daley, Andrew S. Brack, James J. Collins, Chad Cowan, Thorsten M. Schlaeger, Derrick J. Rossi

Cell Stem Cell, 30 September 2010


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