Safe efficient method reported for creating and differentiating human pluripotent stem cells

Boston, Mass. - Researchers at the Immune Disease Institute/Program in Cellular and Molecular Medicine at Children's Hospital Boston report the development of a safe and efficient technology to create human induced pluripotent stem cells, or iPS cells, embryonic-like stem cells created by reprogramming adult cells. Perhaps even more significantly, the researchers demonstrated that their technology could also be used to efficiently steer these stem cells to form cells useful in medicine, such as blood cells, neurons and muscle cells.

The findings, published online September 30 by the journal Cell Stem Cell, are expected to significantly advance the progress of regenerative medicine and disease research.

Current reprogramming protocols for making iPS cells require viruses or DNA to reinstate the stem cell identity, which permanently alters the genome of the cells. The researchers, led by Derrick Rossi, PhD, also affiliated with the Harvard Stem Cell Institute, report a novel technique that uses synthetic modified RNA to generate pluripotent stem cells without irreversibly altering the cells' genetic material. The resulting stem cells very closely recapitulate the functional and molecular properties of human embryonic stem cells, and are generated at much higher efficiencies than standard virus-based techniques.

Importantly, modified RNA can also be used to direct the pluripotent stem cells into cell types that could be used clinically, the researchers show. The difficulty of differentiating iPS cells into clinically useful cell types has been a major obstacle to advancing stem-cell therapies.

"Since our technology can be used to both generate patient-specific stem cells and differentiated cells that can be used therapeutically, we believe it has the potential to become a major enabling technology for the development of cell-based therapies and regenerative medicine," Rossi said.

The 2006 discovery of a way to reprogram fully differentiated adult skin cells into pluripotent stem cells opened up a door to new clinical and research applications of stem cell technology. The stem cells are produced without destroying embryos, and because they are derived from a patient's own cells, cells and tissues generated from them can be transplanted back into patients with no risk of immune rejection.

Getting differentiated cells to regress, or "reprogram" to an embryonic stem cell-like state requires introduction of four key proteins. These proteins are most often introduced using DNA-based viruses, an approach that carries the risk of causing mutations in the reprogrammed cells, which could trigger cancers.

To get around this problem, Rossi and colleagues thought to employ messenger RNA (mRNA) to drive expression of the reprogramming factors since mRNA does not integrate into the cellular DNA. However, they first had to overcome an obstacle: when mRNA was introduced into cells, the cells' natural defense mechanisms interpreted this as a viral infection, and responded with a potent anti-viral reaction that destroyed the RNA and killed the cells. To avoid triggering this anti-viral response, the investigators spent more than a year developing synthetic, chemically modified RNAs that, when introduced into cells, escapes detection by this anti-viral defense system. This permitted the modified mRNA to drive protein expression effectively for days and weeks in human cells without adverse affects on the cells.

The researchers then put their method to the test, treating cells derived from human skin with a cocktail of modified mRNAs encoding the four major reprogramming proteins. With daily treatment, the cells reverted to a pluripotent state similar to human embryonic stem cells. Not only were the cells free of DNA integrations, but the reprogramming process was completed in about half the time required for standard virus-based techniques, and was up to 100 times more efficient.

The modified RNA technology was also effective at redirecting stem cells to form other tissue types. Currently, scientists attempt to coax iPS cells to differentiate to clinically useful cell types by changing their external environment. The new work shows, however, that the addition of a modified RNA encoding a factor important for muscle differentiation directly into the stem cells results in efficient generation of functional muscle cells. This provides a proof of concept that the RNA method could be used to generate patient-specific cells of various types for use in regenerative therapies.

"If tissue engineering is to progress to the clinic, there is a pressing need for efficient, non-mutagenic strategies to redirect cell fate," Rossi said. "Our results show that this novel RNA technology can be used to generate patient-specific pluripotent stem cells, and can likewise be harnessed to direct the fate of such stem cells towards specialized cell types that have the potential to be used clinically."

Rossi notes that their technology has potential reaching far beyond the stem-cell field. The modified mRNAs can be used to boost production of any needed protein in a cell, and therefore could be potentially utilized for treating any genetic disease in which a protein is missing, deficient or defective, such as cystic fibrosis. Whereas RNA interference (RNAi) technology is widely used to inhibit gene activity and protein production, a safe reverse technology hasn't existed until now. Rossi thinks the modified mRNA technology represents this missing technology and will thus be adopted by many labs. He has patented his findings and recently formed a company called ModeRNA Therapeutics that is dedicated to translation of these discoveries into clinical use.

Bess Andrews

Children's Hospital Boston is home to the world's largest research enterprise based at a pediatric medical center, where its discoveries have benefited both children and adults since 1869. More than 1,100 scientists, including nine members of the National Academy of Sciences, 12 members of the Institute of Medicine and 13 members of the Howard Hughes Medical Institute comprise Children's research community. Founded as a 20-bed hospital for children, Children's Hospital Boston today is a 392-bed comprehensive center for pediatric and adolescent health care grounded in the values of excellence in patient care and sensitivity to the complex needs and diversity of children and families. Children's also is the primary pediatric teaching affiliate of Harvard Medical School. For more information about the hospital and its research visit:

Founded in 1953, the Program in Molecular and Cellular Medicine and the Immune Disease Institute (PCMM/IDI) is a non-profit research institution in Boston, MA, recognized worldwide for its discoveries that increase the body's ability to fight disease and to heal. With the aim of increasing collaborations and scientific synergies, Immune Disease Institute and Children's Hospital Boston have entered into an affiliation whereby IDI joins seven other interdisciplinary programs as the Program in Cellular and Molecular Medicine. PCMM/IDI is also academically affiliated with Harvard Medical School, and its investigators hold appointments in departments of the medical school. The breakthroughs of PCMM/IDI scientists are greatly increasing our understanding of the influence of immune defense and inflammation on biomedical discovery, healthcare, and disease management. to learn more.

**** Reference:
Highly efficient reprogramming to pluripotency and directed differentiation of human cells using synthetic modified mRNA Cell Stem Cell, October 2010, Epub Sept 30.

Luigi Warren1,12, Philip D. Manos2,3,12, Tim Ahfeldt4,11, Yuin-Han Loh2,5, Hu Li8, Frank Lau4,6, Wataru Ebina1, Pankaj Mandal1, Zachary D. Smith7, Alexander Meissner7, George Q. Daley2,3,5,9, Andrew S. Brack10, James J. Collins8, Chad Cowan4,6, Thorsten M. Schlaeger2,3, Derrick J. Rossi1

1. Immune Disease Institute, Program in Cellular and Molecular Medicine, Children's Hospital Boston, Harvard Stem Cell Institute, and the Department of Pathology, Harvard Medical School, Boston, MA 02115, USA

2. Division of Pediatric Hematology/Oncology, Children's Hospital Boston and Dana-Farber Cancer Institute, Boston, MA, USA

3. Stem Cell Program, Children's Hospital Boston, Boston, MA, USA

4. Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Massachusetts General Hospital Center for Regenerative Medicine, 185 Cambridge Street, Boston, MA 02114, USA

5. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA

6. Stowers Medical Institute, 185 Cambridge Street, Boston, MA 02114, USA

7. Department of Stem Cell and Regenerative Biology, Harvard University and Harvard Stem Cell Institute, Cambridge, MA 02138, USA, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA

8. Howard Hughes Medical Institute, Department of Biomedical Engineering and Center for BioDynamics, Boston University, Boston, MA 02215, USA, Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA

9. Harvard Stem Cell Institute, Cambridge, MA, USA, Howard Hughes Medical Institute at Children's Hospital Boston, Boston, MA, USA, Division of Hematology/Oncology, Brigham and Women's Hospital, Boston, MA, USA, Manton Center for Orphan Disease Research, Children's Hospital Boston, Boston, MA, USA

10. Center of Regenerative Medicine, Massachusetts General Hospital, 185 Cambridge Street, MGH Charles River Plaza North, Room 4232, Boston, MA 02114-2790, USA

11. Department of Biochemistry and Molecular Biology II: Molecular Cell Biology, University Medical Center Hamburg-Eppendorf, Hamburg 20246, Germany

12. These authors contributed equally to this work

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