For the first time, scientists entirely reprogram human skin cells to iPSCs using CRISPR

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CRISPR iPSC colony of human skin cells showing expression of SOX2 and TRA-1-60, markers of human embryonic pluripotent stem cells

Back in 2012, Shinya Yamanaka was awarded the Nobel Prize in Physiology or Medicine for his group’s identification of “Yamanaka Factors,” a group of genes that are capable of turning ordinary skin cells into induced pluripotentent stem cells (iPSCs) which have the ability to become any type of cell within the body. Discovery of iPSCs was, and has been, groundbreaking because it not only allows for unprecedented avenues to study human disease, but also has implications for using a patient’s own cells to treat a wide variety of diseases.

Recently, Timo Otonkoski’s group at the University of Helsinki along with Juha Kere’s group at the Karolinska Institutet and King’s College, London have found a way to program iPSCs from skin cells using CRISPR, a gene editing technology. Their approach allows for the induction, or turning on of iPSCs using the cells own DNA, instead of introducing the previously identified Yamanka Factors into cells of interest.

As detailed in their study, published in the journal Nature Communications, this is the first instance of mature human cells being completely reprogrammed into pluripotent cells using only CRISPR. Instead of using the canonical CRISPR system that allows the CAS9 protein (an enzyme that is able to cut DNA, thus rendering a gene of interest dysfunctional) to mutate any gene of interest, this group used a modified version of the CAS9 protein, which allows them to turn on or off the gene that CAS9 is targeted to.

The robustness of their approach lies in the researcher’s identification of a DNA sequence that is commonly found near genes involved in embryonic development. As CAS9 needs to be guided to genes of interest to do its job, identification of this common motif allows multiple genes associated with pluripotency to be activated in mature human skin cells, and greatly increased the efficiency and effectiveness of this approach.

In a press release, Dr. Otonkoski further highlights the novelty and viability of this approach:

“…Reprogramming based on activation of endogenous genes rather than overexpression of transgenes is…theoretically a more physiological way of controlling cell fate and may result in more normal cells…”

 

New Regenerative Liver Cells Identified

It’s common knowledge that your liver is a champion when it comes to regeneration. It’s actually one of the few internal organs in the human body that can robustly regenerate itself after injury. Other organs such as the heart and lungs do not have the same regenerative response and instead generate scar tissue to protect the injured area. Liver regeneration is very important to human health as the liver conducts many fundamental processes such as making proteins, breaking down toxic substances, and making new chemicals required to digest your food.

The human liver.

The human liver

Over the years, scientists have suggested multiple theories for why the liver has this amazing regenerative capacity. What’s known for sure is that mature hepatocytes (the main cell type in the liver) will respond to injury by dividing and proliferating to make more hepatocytes. In this way, the liver can regrow up to 70% of itself within a matter of a few weeks. Pretty amazing right?

So what is the source of these regenerative hepatocytes? It was originally thought that adult liver stem cells (called oval cells) were the source, but this theory has been disproved in the past few years. The answer to this million-dollar question, however, likely comes from a study published last week in the journal Cell.

Hybrid hepatocytes (shown in green) divide and regenerate the liver in response to injury. (Image source: Font-Burgada et al., 2015)

Hybrid hepatocytes (green) divide and regenerate the liver in response to injury. (Image source: Font-Burgada et al., 2015)

A group at UCSD led by Dr. Michael Karin reported a new population of liver cells called “hybrid hepatocytes”. These cells were discovered in an area of the healthy liver called the portal triad. Using mouse models, the CIRM-funded group found that hybrid hepatocytes respond to chemical-induced injury by massively dividing to replace damaged or lost liver tissue. When they took a closer look at these newly-identified cells, they found that hybrid hepatocytes were very similar to normal hepatocytes but differed slightly with respect to the types of liver genes that they expressed.

A common concern associated with regenerative tissue and cells is the development of cancer. Actively dividing cells in the liver can acquire genetic mutations that can cause hepatocellular carcinoma, a common form of liver cancer.

What makes this group’s discovery so exciting is that they found evidence that hybrid hepatocytes do not cause cancer in mice. They showed this by transplanting a population of hybrid hepatocytes into multiple mouse models of liver cancer. When they dissected the liver tumors from these mice, none of the transplanted hybrid cells were present. They concluded that hybrid hepatocytes are robust and efficient at regenerating the liver in response to injury, and that they are a safe and non-cancer causing source of regenerating liver cells.

Currently, liver transplantation is the only therapy for end-stage liver diseases (often caused by cirrhosis or hepatitis) and aggressive forms of liver cancer. Patients receiving liver transplants from donors have a good chance of survival, however donated livers are in short supply, and patients who actually get liver transplants have to take immunosuppressant drugs for the rest of their lives. Stem cell-derived liver tissue, either from embryonic or induced pluripotent stem cells (iPSC), has been proposed as an alternative source of transplantable liver tissue. However, safety of iPSC-derived tissue for clinical applications is still being addressed due to the potential risk of tumor formation caused by iPSCs that haven’t fully matured.

This study gives hope to the future of cell-based therapies for liver disease and avoids the current hurdles associated with iPSC-based therapy. In a press release from UCSD, Dr. Karin succinctly summarized the implications of their findings.

“Hybrid hepatocytes represent not only the most effective way to repair a diseased liver, but also the safest way to prevent fatal liver failure by cell transplantation.”

This exciting and potentially game-changing research was supported by CIRM funding. The first author, Dr. Joan Font-Burgada, was a CIRM postdoctoral scholar from 2012-2014. He reached out to CIRM regarding his publication and provided the following feedback:

CIRM Postdoctoral Fellow Jean Font-Burgada

CIRM postdoctoral scholar Joan Font-Burgada

“I’m excited to let you know that work CIRM funded through the training program will be published in Cell. I would like to express my most sincere gratitude for the opportunity I was given. I am convinced that without CIRM support, I could not have finished my project. Not only the training was excellent but the resources I was offered allowed me to work with enough independence to explore new avenues in my project that finally ended up in this publication.”

 

We at CIRM are always thrilled and proud to hear about these success stories. More importantly, we value feedback from our grantees on how our funding and training has supported their science and helped them achieve their goals. Our mission is to develop stem cell therapies for patients with unmet medical needs, and studies such as this one are an encouraging sign that we are making progress towards to achieving this goal.


Related links:

UCSD Press Release

CIRM Spotlight on Liver Disease Research

CIRM Spotlight on Living with Liver Disease

Mutation Morphs Mitochondria in Models of Parkinson’s Disease, CIRM-Funded Study Finds

There is no singular cause of Parkinson’s disease, but many—making this disease so difficult to understand and, as a result, treat. But now, researchers at the Buck Institute for Research on Aging have tracked down precisely how a genetic change, or mutation, can lead to a common form of the disease. The results, published last week in the journal Stem Cell Reports, point to new and improved strategies at tackling the underlying processes that lead to Parkinson’s.

Mitochondria from iPSC-derived neurons. On the left is a neuron derived from a healthy individual, while the image on the right shows a neuron derived from someone with the Park2 mutation, the most common mutation in Parkinson's disease (Credit: Akos Gerencser)

Mitochondria from iPSC-derived neurons. On the left is a neuron derived from a healthy individual, while the image on the right shows a neuron derived from someone with the Park2 mutation, the most common mutation in Parkinson’s disease (Credit: Akos Gerencser)

The debilitating symptoms of Parkinson’s—most notably stiffness and tremors that progress over time, making it difficult for patients to walk, write or perform other simple tasks—can in large part be linked to the death of neurons that secrete the hormone dopamine. Studies involving fruit flies in the lab had identified mitochondria, cellular ‘workhorses’ that churn out energy, as a key factor in neuronal death. But this hypothesis had not been tested using human cells.

Now, scientists at the Buck Institute have replicated the process in human cells, with the help of stem cells derived from patients suffering from Parkinson’s, a technique called induced pluripotent stem cell technology, or iPSC technology. These newly developed neurons exactly mimic the disease at the cellular level. This so-called ‘disease in a dish’ is one of the most promising applications of stem cell technology.

“If we can find existing drugs or develop new ones that prevent damage to the mitochondria we would have a potential treatment for PD,” said Dr. Xianmin Zeng, the study’s senior author, in a press release.

And by using this technology, the Buck Institute team confirmed that the same process that occurred in fruit fly cells also occurred in human cells. Specifically, the team found that a particular mutation in these cells, called Park2, altered both the structure and function of mitochondria inside each cell, setting off a chain reaction that leads to the neurons’ inability to produce dopamine and, ultimately, the death of the neuron itself.

This study, which was funded in part by a grant from CIRM, could be critical in the search for a cure for a disease that, as of yet, has none. Current treatment regimens aimed at slowing or reducing symptoms have had some success, but most begin to fail overtime—or come with significant negative side effects. The hope, says Zeng, is that iPSC technology can be the key to fast-tracking promising drugs that can actually target the disease’s underlying causes, and not just their overt symptoms. Hear more from Dr. Xianmin Zeng as she answers your questions about Parkinson’s disease and stem cell research:

DISCUSSing iPSC Derivation

Geoff Lomax is CIRM’s Senior Officer for Medical and Ethical Standards. He has been working in the implementation of CIRM’s iPSC Banking Program.

The ability to create high-quality stem cell lines depends, in part, on the generosity of donors. For example, CIRM is sponsoring an induced pluripotent stem cell bank (iPSC bank) that will eventually contain 9,000 stem cell lines. Each of these lines will be generated from tissue donated by 3,000 people suffering from known diseases such as Alzheimer’s disease, autism, hepatitis, blindness, heart disease—and many more. You can learn more about this important initiative here.

shutterstock_182164514

In other countries there are similar initiatives like the one sponsored by CIRM.

We also believe that our donors should have accurate information about how their donated materials will be used, so CIRM has developed variety of tools designed to educate donors. For example, each donor must go through a process called “informed consent” where they are told the details of how iPSC’s are derived and preserved in a bank. We discuss this effort here. In the context of the CIRM bank, new donors are being recruited under ethically and scientifically optimal conditions—where they can be fully informed as to how their cells will be used and how their contribution will spur stem cell research.

There are, however, existing libraries of cell and tissues that have inherent scientific value. For example, they may represent a rare or “orphan” disease. Or, they may be essential for tracking the progress of a patient’s disease over time. These collections have also been developed with the consent of the donor or patient, but, at the time of collection, iPSCs may not have even existed. One question that frequently arises is: can these cells be used for iPSC derivation, research and banking? It is not an abstract concern; CIRM and others often get questions about the adequacy of donor consent for precisely this purpose.

In 2013, CIRM, the NIH and the International Stem Cell Forum (ISCF)/McGill University formed the DISCUSS Project (Deriving Induced Stem Cells Using Stored Specimens) to engage the boarder research community on this issue. Rosario Isasi, a project collaborator from ISCF/McGill University, said that her research tells us that investigators around the world are asking the same questions about use of existing cell lines. To help inform researchers, we started by publishing a report on this very subject. The report included nine points to consider when answering the question of whether existing cell libraries can be used for iPSC research.

We followed this initial effort with a series of meetings and workshops to get reactions to our proposed points to consider. The process culminated with a workshop in March where researchers from around world provided recommendations to the DISCUSS team. Sara Hull, a project collaborator from the NIH, noted that the international perspectives were key to producing a greatly improved product. A major workshop theme was the importance of having an effective management system in place, making sure that the cells are used in a way that is consistent with the donor consent. Participants described a number of specific mechanisms that should be used by the research community to ensure cells are used appropriately. Participants emphasized that having effective systems in place to manage cells and iPSC lines in accordance with donors wishes serves to build trust.

Our workshop report elaborates on specific steps researchers and stem cell banks should take to ensure cell lines are used appropriately. The report also includes a revised set of points to consider based on comments received from meetings and workshops.

The DISCUSS Team looks forward to working with the research community to develop consensus for the responsible use of donated materials in stem cell research.

Geoff Lomax

DISCUSSing iPSC Derivation

Geoff Lomax is CIRM’s Senior Officer for Medical and Ethical Standards. He has been working in the implementation of CIRM’s iPSC Banking Program.

The ability to create high-quality stem cell lines depends, in part, on the generosity of donors. For example, CIRM is sponsoring an induced pluripotent stem cell bank (iPSC bank) that will eventually contain 9,000 stem cell lines. Each of these lines will be generated from tissue donated by 3,000 people suffering from known diseases such as Alzheimer’s disease, autism, hepatitis, blindness, heart disease—and many more. You can learn more about this important initiative here.

shutterstock_182164514

In other countries there are similar initiatives like the one sponsored by CIRM.

We also believe that our donors should have accurate information about how their donated materials will be used, so CIRM has developed variety of tools designed to educate donors. For example, each donor must go through a process called “informed consent” where they are told the details of how iPSC’s are derived and preserved in a bank. We discuss this effort here. In the context of the CIRM bank, new donors are being recruited under ethically and scientifically optimal conditions—where they can be fully informed as to how their cells will be used and how their contribution will spur stem cell research.

There are, however, existing libraries of cell and tissues that have inherent scientific value. For example, they may represent a rare or “orphan” disease. Or, they may be essential for tracking the progress of a patient’s disease over time. These collections have also been developed with the consent of the donor or patient, but, at the time of collection, iPSCs may not have even existed. One question that frequently arises is: can these cells be used for iPSC derivation, research and banking? It is not an abstract concern; CIRM and others often get questions about the adequacy of donor consent for precisely this purpose.

In 2013, CIRM, the NIH and the International Stem Cell Forum (ISCF)/McGill University formed the DISCUSS Project (Deriving Induced Stem Cells Using Stored Specimens) to engage the boarder research community on this issue. Rosario Isasi, a project collaborator from ISCF/McGill University, said that her research tells us that investigators around the world are asking the same questions about use of existing cell lines. To help inform researchers, we started by publishing a report on this very subject. The report included nine points to consider when answering the question of whether existing cell libraries can be used for iPSC research.

We followed this initial effort with a series of meetings and workshops to get reactions to our proposed points to consider. The process culminated with a workshop in March where researchers from around world provided recommendations to the DISCUSS team. Sara Hull, a project collaborator from the NIH, noted that the international perspectives were key to producing a greatly improved product. A major workshop theme was the importance of having an effective management system in place, making sure that the cells are used in a way that is consistent with the donor consent. Participants described a number of specific mechanisms that should be used by the research community to ensure cells are used appropriately. Participants emphasized that having effective systems in place to manage cells and iPSC lines in accordance with donors wishes serves to build trust.

Our workshop report elaborates on specific steps researchers and stem cell banks should take to ensure cell lines are used appropriately. The report also includes a revised set of points to consider based on comments received from meetings and workshops.

The DISCUSS Team looks forward to working with the research community to develop consensus for the responsible use of donated materials in stem cell research.

Geoff Lomax