Thursday, 27 November 2014

iPS Cells Used to Correct Genetic Mutations that Cause Muscular Dystrophy

iPS Cells Used to Correct Genetic Mutations that Cause Muscular Dystrophy
Thursday, 27 November 2014

This image shows immunofluorescence staining
of skeletal cells differentiated from DMD-iPS
cells. Untreated DMD skeletal cells do not
express dystrophin (green) due to the deletion of
exon 44. However, after any of the three
correction strategies are applied to iPS cells,
differentiation into skeletal cells results in normal
dystrophin expression. Scale bar, 50 μm.
Credit: Dr. Akitsu Hotta, Kyoto University.
Researchers at the Center for iPS Cell Research and Application (CiRA), Kyoto University, show that induced pluripotent stem (iPS) cells can be used to correct genetic mutations that cause Duchenne muscular dystrophy (DMD). The research, published in Stem Cell Reports, demonstrates how engineered nucleases, such as TALEN and CRISPR, can be used to edit the genome of iPS cells generated from the skin cells of a DMD patient. The cells were then differentiated into skeletal muscles, in which the mutation responsible for DMD had disappeared.

DMD is a severe muscular degenerative disease caused by a loss-of-function mutation in the dystrophin gene. It inflicts 1 in 3500 boys and normally leads to death by early adulthood. Currently, very little is available in terms of treatment for patients outside palliative care. One option gaining interest is genomic editing by TALEN and CRISPR, which have quickly become invaluable tools in molecular biology. These enzymes allow scientists to cleave genes at specific locations and then modify the remnants to produce a genomic sequence to their liking. However, programmable nucleases are not pristine and often mistakenly edit similar sequences that vary a few base pairs from the target sequence, making them unreliable for clinical use because of the potential for undesired mutations.

For this reason, induced pluripotent stem cells (iPS cells) are ideal models, because they provide researchers an abundance of patient cells on which to test the programmable nucleases and find optimal conditions that minimize off-target modifications. CiRA scientists took advantage of this feature by generating iPS cells from a DMD patient. They used several different TALEN and CRISPR to modify the genome of the iPS cells, which were then differentiated into skeletal muscle cells. In all cases, dystrophin protein expression was convalesced, and in some cases, the dystrophin gene was fully corrected.

One key to the success was the development of a computational protocol that minimized the risk of off-target editing. The team built a database that all possible permutations of sequences up to 16 base pairs long. Among these, they extracted those that only appear once in the human genome, i.e. unique sequences. DMD can be caused by several different mutations; in the case of the patient used in this study, it was the result of the deletion of exon 44. The researchers therefore built a histogram of unique sequences that appeared in a genomic region that contained this exon. They found a stack of unique sequences in exon 45.

to Akitsu Hotta, who headed the project and holds joint positions at CiRA and the Institute for Integrated Cell-Materials Sciences at Kyoto University:

"Nearly half the human genome consists of repeated sequences. So even if we found one unique sequence, a change of one or two base pairs may result in these other repeated sequences, which risks the TALEN or CRISPR editing an incorrect region. To avoid this problem, we sought a region that hit high in the histogram".

With this target, the team considered three strategies to modify the frame-shift mutation of the dystrophin gene: exon skipping by connecting exons 43 and 46 to restore the reading frame, frame shifting by incorporating insertion or deletion (indel) mutations, and exon knock-in by inserting exon 44 before exon 45. All three strategies effectively increased dystrophin synthesis in differentiated skeletal cells, but only the exon knock-in approach recovered the gene to its natural state. Importantly, editing showed very high specificity, suggesting that their computational approach can be used to minimize off-target editing by the programming nucleases.

Moreover, the paper provides a proof-of-principle for using iPS cell technology to treat DMD in combination with TALEN or CRISPR. The group now aims to expand this protocol to other diseases.

First author Lisa Li explains: "We show that TALEN and CRISPR can be used to correct the mutation of the DMD gene. I want to apply the nucleases to correct mutations for other genetic-based diseases like point mutations".

Contact: Akemi Nakamura

Reference:
Precise correction of the DYSTROPHIN gene in Duchenne Muscular Dystrophy patient-derived iPS cells by TALEN and CRISPR-Cas9
Hongmei Lisa Li, Naoko Fujimoto, Noriko Sasakawa, Saya Shirai, Tokiko Ohkame, tetsushi Sakuma, Michihiro tanaka, Naoki Amano, Akira Watanabe, Hidetoshi Sakurai, Takashi Yamamoto, Shinya Yamanaka, and Akitsu Hotta
Stem Cell Reports, November 26, 2014, DOI: http://dx.doi.org/10.1016/j.stemcr.2014.10.013
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Friday, 21 November 2014

WNT Signalling Molecule Crucial to Stem Cell Reprogramming

Surprise finding has implications for wound repair therapies and inhibiting cancer
Friday, 21 November 2014

While investigating a rare genetic disorder, researchers at the University of California, San Diego School of Medicine have discovered that a ubiquitous signalling molecule is crucial to cellular reprogramming, a finding with significant implications for stem cell-based regenerative medicine, wound repair therapies and potential cancer treatments.

The findings are published in the Nov. 20 online issue of Cell Reports.

Karl Willert, PhD, assistant professor in the Department of Cellular and Molecular Medicine, and colleagues were attempting to use induced pluripotent stem cells (iPSC) to create a "disease-in-a-dish" model for focal dermal hypoplasia (FDH), a rare inherited disorder caused by mutations in a gene called PORCN. Study co-authors V. Reid Sutton and Ignatia Van den Veyver at Baylor College of Medicine had published the observation that PORCN mutations underlie FDH in humans in 2007.

FDH is characterized by skin abnormalities such as streaks of very thin skin or different shades, clusters of visible veins and wart like growths. Many individuals with FDH also suffer from hand and foot abnormalities and distinct facial features. The condition is also known as Goltz syndrome after Robert Goltz, who first described it in the 1960s. Goltz spent the last portion of his career as a professor at UC San Diego School of Medicine. He retired in 2004 and passed away earlier this year.

To their surprise, Willert and colleagues discovered that attempts to reprogram FDH fibroblasts or skin cells with the requisite PORCN mutation into iPSCs failed using standard methods, but succeeded when they added WNT proteins - a family of highly conserved signalling molecules that regulate cell-to-cell interactions during embryogenesis.

"WNT signalling is ubiquitous," said Willert.

"Every cell expresses one or more WNT genes and every cell is able to receive WNT signals. Individual cells in a dish can grow and divide without WNT, but in an organism, WNT is critical for cell-cell communication so that cells distinguish themselves from neighbours and thus generate distinct tissues, organs and body parts."

WNT signalling is also critical in limb regeneration (in some organisms) and tissue repair.

"We've shown that WNT signalling is required for cellular reprogramming," said Willert.

"Some of the processes that occur during cellular reprogramming resemble those that occur during regenerative processes and wound repair. For example, limb regeneration in organisms like axolotl and zebrafish require cells at the injury site to de-differentiate (change their function) and then rebuild the damaged tissue. WNT is essential for these amazing regenerative processes."

Willert cautioned that "it would be a stretch to say that activation of WNT signalling will allow us to regenerate limbs," but said WNT activation is likely valuable in assisting tissue repair.

A variety of efforts are already underway exploring how to leverage WNT signalling to promote wound healing, such as speeding bone fracture repairs, and even hair growth.

"That's not really a wound repair process, but WNT is required for hair growth," Willert said.

The caveat, he noted, is that "there's a fine line between repairing tissue and promoting cancer growth." Willert said there are efforts underway to create therapeutics that block WNT signalling as a means to block cancer growth. Earlier this year, for example, Willert and colleagues published findings describing the use of an antibody to disrupt WNT signalling in embryonic stem cells. In cancer cells with mutations in the WNT signalling pathway this antibody may inhibit their growth and development.

Contact: Scott LaFee

Reference:
A Rare Human Syndrome Provides Genetic Evidence that WNT Signaling Is Required for Reprogramming of Fibroblasts to Induced Pluripotent Stem Cells
Jason Ross, Julia Busch, Ellen Mintz, Damian Ng, Alexandra Stanley, David Brafman, V. Reid Sutton, Ignatia Van den Veyver, Karl Willert
Cell Reports, November 20, 2014, DOI: http://dx.doi.org/10.1016/j.celrep.2014.10.049
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Pluripotent Cells Created by Nuclear Transfer Can Prompt Immune Reaction

Pluripotent Cells Created by Nuclear Transfer Can Prompt Immune Reaction
Friday, 21 November 2014

Mouse cells and tissues created through nuclear transfer can be rejected by the body because of a previously unknown immune response to the cell's mitochondria, according to a study in mice by researchers at the Stanford University School of Medicine and colleagues in Germany, England and at MIT.

The findings reveal a likely, but surmountable, hurdle if such therapies are ever used in humans, the researchers said.

Stem cell therapies hold vast potential for repairing organs and treating disease. The greatest hope rests on the potential of pluripotent stem cells, which can become nearly any kind of cell in the body. One method of creating pluripotent stem cells is called somatic cell nuclear transfer, and involves taking the nucleus of an adult cell and injecting it into an egg cell from which the nucleus has been removed.

The promise of the SCNT method is that the nucleus of a patient's skin cell, for example, could be used to create pluripotent cells that might be able to repair a part of that patient's body.

"One attraction of SCNT has always been that the genetic identity of the new pluripotent cell would be the same as the patient's, since the transplanted nucleus carries the patient's DNA," said cardiothoracic surgeon Sonja Schrepfer, MD, PhD, a co-senior author of the study, which will be published online Nov. 20 in Cell Stem Cell.

"The hope has been that this would eliminate the problem of the patient's immune system attacking the pluripotent cells as foreign tissue, which is a problem with most organs and tissues when they are transplanted from one patient to another," added Schrepfer, who is a visiting scholar at Stanford's Cardiovascular Institute. She is also a Heisenberg Professor of the German Research Foundation at the University Heart Center in Hamburg, and at the German Center for Cardiovascular Research.

Possibility of rejection
A dozen years ago, when Irving Weissman, MD, professor of pathology and of developmental biology at Stanford, headed a National Academy of Sciences panel on stem cells, he raised the possibility that the immune system of a patient who received SCNT-derived cells might still react against the cells' mitochondria, which act as the energy factories for the cell and have their own DNA. This reaction could occur because cells created through SCNT contain mitochondria from the egg donor and not from the patient, and therefore could still look like foreign tissue to the recipient's immune system, said Weissman, the other co-senior author of the paper. Weissman is the Virginia and D.K. Ludwig Professor for Clinical Investigation in Cancer Research and the director of the Stanford Institute for Stem Cell Biology and Regenerative Medicine.

That hypothesis was never tested until Schrepfer and her colleagues took up the challenge.

"There was a thought that because the mitochondria were on the inside of the cell, they would not be exposed to the host's immune system," Schrepfer said.

"We found out that this was not the case."

Schrepfer, who heads the Transplant and Stem Cell Immunobiology Laboratory in Hamburg, used cells that were created by transferring the nuclei of adult mouse cells into enucleated eggs cells from genetically different mice. When transplanted back into the nucleus donor strain, the cells were rejected although there were only two single nucleotide substitutions in the mitochondrial DNA of these SCNT-derived cells compared to that of the nucleus donor.

"We were surprised to find that just two small differences in the mitochondrial DNA were enough to cause an immune reaction," she said.

"We didn't do the experiment in humans, but we assume the same sort of reaction could occur," Schrepfer added.

Until recently, researchers were able to perform SCNT in many species, but not in humans. When scientists at the Oregon Health and Science University announced success in performing SCNT with human cells last year, it reignited interest in eventually using the technique for human therapies. Although many stem cell researchers are focused on a different method of creating pluripotent stem cells, called induced pluripotent stem cells, there may be some applications for which SCNT-derived pluripotent cells are better suited.

Handling the reaction
The immunological reactions reported in the new paper will be a consideration if clinicians ever use SCNT-derived stem cells in human therapy, but such reactions should not prevent their use, Weissman said.

"This research informs us of the margin of safety that would be required if, in the distant future, we need to use SCNT to create pluripotent cells to treat someone," he said.

"In that case, clinicians would likely be able to handle the immunological reaction using the immunosuppression methods that are currently available."

In the future, scientists might also lessen the immune reaction by using eggs from someone who is genetically similar to the recipient, such as a mother or sister, Schrepfer added.

Contact: Christopher Vaughan 

Reference:
SCNT-Derived ESCs with Mismatched Mitochondria Trigger an Immune Response in Allogeneic Hosts
Tobias Deuse, Dong Wang, Mandy Stubbendorff, Ryo Itagaki, Antje Grabosch, Laura C. Greaves, Malik Alawi, Anne Grünewald, Xiaomeng Hu, Xiaoqin Hua, Joachim Velden, Hermann Reichenspurner, Robert C. Robbins, Rudolf Jaenisch, Irving L. Weissman, Sonja Schrepfer
Cell Stem Cell, November 20, 2014, DOI: http://dx.doi.org/10.1016/j.stem.2014.11.003
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