Researchers Identify How Heart Stem Cells Orchestrate Regeneration

Exosomes, tiny 'bubbles' secreted by specialized cardiac stem cells, appear to carry 'instructions' that spur heart cells to regenerate following heart attack

Wednesday, 07 May 2014

Investigators at the Cedars-Sinai Heart Institute – whose previous research showed that cardiac stem cell therapy reduces scarring and regenerates healthy tissue after a heart attack in humans – have identified components of those stem cells responsible for the beneficial effects.

In a series of laboratory and lab animal studies, Heart Institute researchers found that exosomes, tiny membrane-enclosed "bubbles" involved in cell-to-cell communication, convey messages that reduce cell death, promote growth of new heart muscle cells and encourage the development of healthy blood vessels.

"Exosomes were first described in the mid-1980s, but we only now are beginning to appreciate their potential as therapeutic agents. We have found that exosomes and the cargo they contain are crucial mediators of stem cell-based heart regeneration, and we believe this might lead to an even more refined therapy using the 'active ingredient' instead of the entire stem cell," said Eduardo Marbán, MD, PhD, director of the Cedars-Sinai Heart Institute and a pioneer in developing investigational cardiac stem cell treatments.

"The concept of exosome therapy is interesting because it could potentially shift our strategy from living-cell transplantation to the use of a non-living agent," he added.

"Stem cells must be carefully preserved to keep them alive and functioning until the time of transplant, and there are some risks involved in cell transplantation. In contrast, exosome therapy may be safer and simpler and based on a product with a longer shelf life."

In lab experiments, the researchers isolated exosomes from specialized human cardiac stem cells and found that exosomes alone had the same beneficial effects as stem cells. Exosomes also produced the same post-heart attack benefits in mice, decreasing scar size, increasing healthy heart tissue and reducing levels of chemicals that lead to inflammation. Even when exosomes were injected in mice after heart attack scars were well-established, and traditionally viewed as "irreversible," they brought about multiple structural and functional benefits.

Exosomes transport small pieces of genetic material, called microRNAs, which enable cells to communicate with neighbouring cells to change their behaviour. The researchers pinpointed one such microRNA – one that is especially plentiful in cardiac stem cell exosomes – as responsible for some of the benefits. It is likely, they believe, that this and other microRNAs in the exosomes work together to produce the regenerative effects.

"The exosomes appear to contain the signalling information needed to regenerate healthy heart tissue, they are naturally able to permeate cells, and they have a coating that protects their payloads from degradation as they shuttle from cell to cell," said Marbán, senior author of an article in the May 6, 2014 Stem Cell Reports.

"Injecting exosomes derived from specialized cardiac stem cells may be an attractive alternative to the transplantation of living cells."

Marbán and his clinical and research teams in 2009 performed the first procedure in which a heart attack patient's heart tissue was used to grow specialized stem cells that were injected back into the heart. In 2012, they reported results of a clinical trial that found significant reduction in the size of heart attack-caused scars in patients who underwent the experimental stem cell procedure, compared to others who did not.

They also published findings from an animal study showing that the effect of stem cell therapy following heart attack is indirect – the stem cells themselves do not survive long after being placed in the heart, but they cause enduring effects by stimulating the rapid growth of surviving heart tissue and attracting stem cells already in the heart, which mature into functional heart cells.

The new study sheds light on the underlying mechanisms, crediting stem cell exosomes and the communications cargo they carry for orchestrating regeneration to repair heart attack damage.

The process to grow cardiac-derived stem cells was developed earlier by Marbán when he was on the faculty of Johns Hopkins University. The university has filed for a patent on that intellectual property and has licensed it to Capricor Inc., a biotechnology company in which Marbán is a founder and equity holder. Cedars-Sinai has filed for a patent for the exosome discovery and has licensed it to Capricor. The company provided no funding for this study.

Source: Cedars-Sinai Medical Center 

Contact: Sally Stewart

Reference:

Exosomes as Critical Agents of Cardiac Regeneration Triggered by Cell Therapy

Ahmed Gamal-Eldin Ibrahim, Ke Cheng, Eduardo Marbán

Stem Cell Reports, 6 May 2014, Volume 2, Issue 5, p606–619

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One Step Closer to Cell Reprogramming

One Step Closer to Cell Reprogramming

Wednesday, 07 May 2014

In 2012, John B. Gurdon and Shinya Yamanaka were awarded the Nobel Prize in medicine for discovering that adult cells can be reprogrammed into pluripotent ones (iPS); the cells obtained are capable of behaving in a similar way to embryonic stem cells, and hence have enormous potential for regenerative medicine.

Cells with activated Wnt can no longer be

reprogrammed (in green) are located on the

periphery; cells that can be reprogrammed are

aggregated and can be seen in the centre of the

image (in red). Credit: CRG.

However, although there are many research groups around the world studying this process, it is still not completely understood, it is not totally efficient, and it is not safe enough to be used as the basis for a new cell therapy.

Now, researchers at the Centre for Genomic Regulation (CRG) in Barcelona have taken a very important step towards understanding cell reprogramming and its efficiency: they have discovered the key role of the Wnt signalling pathway in transforming adult cells into iPS cells.

"Generally, transcription factors are used to try to increase or decrease the cell reprogramming process. We have discovered that we can increase the efficiency of the process by inhibiting the Wnt route", explains Francesco Aulicino, a PhD student in the Reprogramming and Regeneration group, led by Maria Pia Cosma and co-author of the study that has just been published in Stem Cell Reports.

The Wnt signalling pathway is a series of biochemical reactions that are produced in cells. In frogs or lizards, for example, these reactions are those that allow their extremities to regenerate if the animal suffers an injury. Although in general, humans and mammals have lost this regenerative capacity, the Wnt pathway is involved in numerous processes during embryonic development and cell fusion, as it is in reprogramming.

The researchers have studied how the Wnt route behaves throughout the entire process of transforming cells into iPS cells, which usually lasts two weeks. It is a very dynamic process that produces oscillations from the pathway, which is not active all the time.

"We have seen that there are two phases and that in each one of them, Wnt fulfils a different function. And we have shown that by inhibiting it at the beginning of the process and activating it at the end we can increase the efficiency of reprogramming and obtain a larger number of pluripotent cells", indicates Ilda Theka, also a PhD student in Pia Cosma's group and a co-author of the article.

To artificially control the pathway, the group has employed a chemical molecule, Iwp2, which is a Wnt secretion inhibitor that does not permanently alter the cells, something which other research into reprogramming using different factors has still has not been able to achieve.

They have also seen that the exact moment when the Wnt pathway is activated is crucial. Doing it too early, makes the cells begin to differentiate, for example into neurones or endodermal cells, and they are not reprogrammed.

"It is a very important and an innovative advance in the field of cell reprogramming, because until now this was a very inefficient process. There are many groups trying to understand the mechanism by which adult cells become pluripotent, and what blocks that process and makes only a small percentage of cells end up being reprogrammed. We are providing information on why it happens", says Theka.

The work opens the way to new advances in regenerative medicine and sheds light on certain types of tumours involving the Wnt pathway. Other labs are also working on ways to increase efficiency when inducing pluripotency in these cells. This is the case of the Haematopoietic Stem Cells, Transdifferentiation and Reprogramming laboratory, led by Thomas Graf, where they work on induced pluripotent stem cells (iPS).

Source: Center for Genomic Regulation 

Contact: Juan Manuel Sarasua

Reference:

Temporal Perturbation of the Wnt Signaling Pathway in the Control of Cell Reprogramming Is Modulated by TCF1

Francesco Aulicino, Ilda Theka, Luigi Ombrato, Frederic Lluis, Maria Pia Cosma

Stem Cell Reports, 6 May 2014, Volume 2, Issue 5, p707–720

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Ability to Isolate and Grow Breast Tissue Stem Cells Could Speed Cancer Research

Salk scientists find two key proteins that regulate the growth of mammary stem cells and could contribute to breast cancer 

Monday, 05 May 2014

By carefully controlling the levels of two proteins, researchers at the Salk Institute have discovered how to keep mammary stem cells – those that can form breast tissue – alive and functioning in the lab. The new ability to propagate mammary stem cells is allowing them to study both breast development and the formation of breast cancers.

Peter C. Gray, Benjamin T. Spike and Geoffrey

M. Wahl. Credit: Courtesy of the Salk Institute

for Biological Studies. 

"What we've shown is that we can take these cells out of a mouse and study them and regulate them in the laboratory by providing them with a specific factor," says Peter C. Gray, a staff scientist in Salk's Clayton Foundation Laboratories for Peptide Biology, who collaborated on the new work with Benjamin T. Spike, a senior research associate in the laboratory of Salk Professor Geoffrey M. Wahl.

The results of the study were published in the April 8th, 2014 issue of the journal Stem Cell Reports.

Mammary stem cells can give rise to new breast cells during foetal development, adolescence or lactation and may also play a role in breast cancer, so they represent a highly promising avenue for breast cancer research. But isolating the stem cells and maintaining them in the lab to study has been difficult.

"There was a lot of prior work demonstrating that mammary-specific stem cells exist, but it was virtually impossible to isolate them in numbers from an adult," says Spike.

"But we previously found we could turn to early development, when the stem cells are present in higher proportions."

When scientists add CRIPTO to a population of

breast stem cells, they retain their ability to

produce more stem cells, keeping the population

constant. But when CRIPTO's action is blocked

with the molecule ALK4, the cells differentiate

into mature cells and the population of stem cells

shrinks. Credit: Salk Institute for Biological

Studies. 

When the researchers used foetal breast tissue rather than adult tissue from mice, they were able to pinpoint which cells were stem cells but the cells would rapidly change when grown in a dish. A defining property of all stem cells is that when they divide into two new cells, they can form both stem cells and differentiated cells (cells on their way to becoming a specific type of tissue).

Spike and Gray grew the mammary stem cells in culture dishes and stained them so that new stem cells appeared a different colour from differentiated mammary cells. Then, they began testing the effects of two proteins – known as CRIPTO and GRP78 – that play significant roles in both stem cell biology and embryonic development.

"In normal conditions, we first see the cells as yellow – the combination of red and green within a single cell – then later see cells that are either red or green, showing that our cells had the capacity to make two different types of mature cells," says Spike.

"But then when we do the experiment again and start changing protein levels, the ratio of these cells becomes very different."

Isolated foetal mammary cells show high levels

of CRIPTO (green) and GRP78 (red), which have

been found to help control the differentiation of

mammary stem cells. Credit: Salk Institute for

Biological Studies. 

The researchers found that when they blocked CRIPTO, the cells mostly formed differentiated cells instead of new stem cells. Over time, this stem cell population shrank since they weren't repopulating themselves. When they instead boosted levels of CRIPTO, the stem cell colony grew as new stem cells were produced more often than differentiated cells.

In studies in mice, the scientists also found that CRIPTO helped the animals form new mammary tissues, which led the team to hypothesize that CRIPTO may be produced by nearby cells in the fat to spur the growth of breast tissue.

In a previous study, Gray's group had discovered that the protein GRP78 binds CRIPTO on the surface of cells and regulates CRIPTO function. This prompted the scientists to test whether GRP78 had an effect on the mammary stem cells. As they suspected, when cells lacked GRP78 on their surfaces, they didn't respond to CRIPTO.

Both CRIPTO and GRP78 have been implicated in cancers, including breast cancer and lung cancers. Scientists think high levels of either protein could encourage tumour growth using similar pathways that they use to spur breast tissue growth. With the new ability to isolate and sustain mammary stem cells, Spike and Gray hope they can uncover details on exactly what cellular programs CRIPTO and GRP78 activate. Understanding this in stem cells could further understanding on how these proteins are involved in tumour growth.

Additionally the researchers think that targeting CRIPTO and GRP78 – which are ideal drug targets since they are present outside of cells – could halt or slow cancer growth. 

"It's looking more and more like what's required to target cancer is to have many therapeutics hitting different pathways," says Gray.

"We think targeting CRIPTO and GRP78 could be a unique way of supplementing existing treatment modalities by targeting stem cell-like cells in cancer."

Source: Salk Institute

Contact: Chris Emery

Reference:

CRIPTO/GRP78 Signaling Maintains Fetal and Adult Mammary Stem Cells Ex Vivo

Benjamin T. Spike, Jonathan A. Kelber, Evan Booker, Madhuri Kalathur, Rose Rodewald, Julia Lipianskaya, Justin La, Marielle He, Tracy Wright, Richard Klemke, Geoffrey M. Wahl, Peter C. Gray

Stem Cell Report, 8 April 2014, Volume 2, Issue 4, p427–439

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