How did you first become involved in the study of cardiovascular disease (CVD)?

It was when I started my PhD in 2007 at the Australian Institute for Bioengineering and Nanotechnology at the University of Queensland; I was interested in cardiac tissue engineering and was lucky enough to have a supervisor, Justin Cooper-White, who let me pursue that interest.

What is currently known about the regenerative capacity of cardiac tissue?

Cardiac tissue has an extremely low turnover compared to other tissues in the body (eg. blood cells and gut tissues are replaced many times during a lifetime). Current estimates are that less than 1 per cent of heart muscle cells (cardiomyocytes) are replaced each year in an adult. Therefore, following injury such as a heart attack (where 1 billion cardiomyocytes can die), the lost cells are not replaced. This leads to disease and remodelling processes to compensate for the lost contractile function, which gradually leads to heart failure.

Interestingly, lower vertebrates such as zebrafish can fully regenerate their hearts

following injury. Cardiomyocytes replicate themselves, rather than being generated from a stem cell population as is the case for other tissues. Recently, my co-Lab Head, Dr Enzo Porrello, discovered a very similar regenerative process in the injured hearts of newborn mice, which only lasts for a few days after birth. We are studying what causes this loss of regenerative capacity in newborn mammals and in 3D human cardiac tissues. In the future, we hope to restore the regenerative response in patients with heart failure to potentially prevent them from developing the condition.

In 2011, the German Cardiovascular Society awarded you a postdoctoral fellowship. What were the key objectives and outcomes of your research at this time?

My research focused on producing cardiac tissues for preclinical studies. This involved the development of robust and efficient protocols to direct the differentiation of human pluripotent stem cells into human cardiac cells and to fabricate cardiac tissue from those cells. Subsequently, we generated induced pluripotent stem cells from rhesus monkeys and produced cardiac tissues from these cells, with the goal to apply them in immuno-compatible preclinical studies in rhesus monkeys.

Following this, you were awarded the National Health and Medical Research Council Peter Doherty Fellowship to further develop this research. How have your studies progressed so far?

We are currently setting up a drug discovery pipeline in our lab, which will allow us to screen for the most effective regenerative treatments or treatments that limit the cell death caused by heart attack in human cardiac tissues. We will test the putative treatments in animal models following heart attack, developed by Dr Enzo Porrello, to prove they work in whole organisms.

To what extent do the 3D human heart tissues created in your laboratory accurately replicate the human heart?

Our 3D cardiac tissues recapitulate many aspects of native hearts, forming organised, elongated, striated muscle (see the image above). Additionally, they respond to physiological and pathological stimuli in a similar manner to native hearts. However, while they display improved maturity over 2D cultures, they are not yet as mature as adult heart tissues. We are currently determining the factors that drive the maturation process into an adult-like state.

Finally, how important is collaboration to your line of research?

Collaboration is essential in today’s scientific environment. In our field, many of the leading journals now require large, innovative studies with findings confirmed using multiple techniques and assays. In many cases, it is not feasible for a lab to develop expertise in all techniques, therefore collaboration is required. This greatly improves the quality and impact of the science. However, it is important that funding bodies and governments consider policies to enhance collaboration over competition.

We collaborate with Professor Walter Thomas, looking at the functional role of ‘taste’ receptors in human heart tissue, and are currently working with Dr David Elliott at the Murdoch Children’s Research Institute in Melbourne, Australia. Elliott’s lab has developed very useful stem cell lines, which have fluorescent reporters in cardiac muscle cells enabling us to track, purify and study cardiac cells. We also collaborate with Professor Rob Parton, who has developed advanced electron microscopy imaging techniques, allowing us to look at the structure of our engineered cardiac tissues in 3D with unprecedented spatial resolution.

Dr James Hudson is working to develop regenerative treatments for the heart, ultimately aiming to find a cure for heart failure patients. Here, he discusses why the heart – a tissue with a low turnover of cells – requires such a treatment and his progressive work to create 3D human heart tissue in the lab

Healing hearts

HUMAN CARDIAC CELLS CULTURED USING 3D TISSUE ENGINEERING

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HUMAN CARDIAC CELLS CULTURED IN 2D

The route to heart

regeneration

University of Queensland researchers are unravelling the molecular mechanisms that drive the regeneration of newborn heart tissue, an ability lost in the adult heart. Searching for new therapeutic targets, their work could prevent heart failure, a common complication after a heart attack

CERTAIN PARTS OF the body have the ability to regenerate; the liver famously can even regenerate after resection, as its cells have a seemingly unlimited capacity for division. However, the heart is not a member of this group as it is unable to renew its lost cells. The limited regenerative capacity of the adult heart becomes particularly significant following a heart attack, after which a series of remodelling processes take place, leading to loss of function and ultimately heart failure. As the leading cause of death worldwide, there is a clear need to find new ways to treat cardiovascular disease.

Steering efforts to stimulate cardiac regeneration, Dr James Hudson co-heads (together with Dr Enzo Porrello) the Cardiac Regeneration Laboratory at the University of Queensland in Australia. At the basic level, the laboratory aims to understand the molecular mechanisms underlying the development and regeneration of heart tissue. In doing so, they made an amazing discovery – unlike the adult heart, the newborn heart can regenerate itself after injury. In newborn mice, the researchers found that the heart has a short-lived potential for regeneration, lost soon after birth. Like the zebrafish, the neonatal mouse heart is able to regenerate itself after an astounding range of cardiac insults, including heart attack, freezing, and even surgical amputation.

Following this striking finding, the team is now working to identify the molecular level differences in the newborn heart that enable

it to regenerate. Although it seems to be driven by the proliferation of resident cardiac muscle cells (cardiomyocytes), the detailed mechanisms underlying this remain poorly understood. Using a range of approaches including molecular genetic studies in mice and human bioengineered heart muscle as a model of human heart tissue, Hudson aims to find a way to replenish the cardiomyocytes lost following a heart attack. By enabling the heart to heal itself, he intends to prevent heart failure.

DIRECTED DIFFERENTIATION

In order to produce human bioengineered heart muscle, the lab is exploiting recent advances in tissue engineering and the science of pluripotent stem cells, which can differentiate into any cell in the body. “Pluripotent stem cells are now widely used to produce different human cell types. Research over the past 15 years has facilitated the development of protocols able to produce millions of high quality cells with relative ease,” Hudson explains. Indeed, the recent development of advanced protocols in directing pluripotent stem cells to become cardiac cells has made their use popular worldwide.

The standard methodology to culture cells though, culturing in 2D (on flat, plastic dishes), leaves much room for improvement. Culturing cardiomyocytes taken from animal hearts in a 2D culture does not accurately maintain their phenotype, instead leading to

de-differentiation, immature gene expression profiles and an overgrowth of non-muscle cells. Furthermore, they cannot be used to measure the force of contraction, an important gauge of heart function. Put simply, they are poor representations of cardiomyocytes in intact hearts. However, there is an alternative. “Using 3D tissue engineering approaches, these caveats can be overcome to provide a much more accurate representation of the human heart,” Hudson states.

3D tissue engineering approaches more closely mimic natural tissues, allowing the cells to align and electrically and mechanically couple. Using this approach to culture pluripotent stem cell-derived cardiomyocytes leads to more mature cardiac cultures, which display many morphological and functional properties similar to the native heart.

CARDIAC TISSUE ENGINEERING

Before cardiomyocytes can be produced, stem cells must be obtained. In Hudson’s lab, researchers use either human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). ESCs are derived directly from blastocysts (an embryo that has developed for five to six days after fertilisation). These can be taken from excess human fertilised eggs discarded by IVF clinics, but can be a controversial option in some countries. The other method is based on reprogramming cells. By introducing stem cell factors into human

DR JAMES HUDSON

22 INTERNATIONAL INNOVATION

The Cardiac Regeneration

Laboratory is now able to

produce an enormous 50

million cardiac cells every week,

enabling the reliable fabrication

of human cardiac tissues

UNRAVELLING THE MOLECULAR MECHANISMS DRIVING CARDIAC REGENERATION

OBJECTIVES

To understand the molecular mechanisms that underpin cardiac regeneration using human bioengineered heart tissue.

KEY COLLABORATORS

Professor Walter Thomas, University of Queensland, Australia

Dr David Elliott, Murdoch Children’s Research Institute, Australia

Professor Rob Parton, University of Queensland, Australia

FUNDING

National Health and Medical Research Council (NHMRC)

CONTACT

Dr James HudsonCo-Head, Cardiac Regeneration Laboratory

Room 511Sir William MacGregor Building (64)St LuciaThe University of QueenslandAustralia

T +61 7 336 52957E j.hudson@uq.edu.au

www.cardiacregenerationlab.com

www.uq.edu.au/sbms/staff/james-hudson

DR JAMES HUDSON completed a PhD in 2011 at the Australian Institute for Bioengineering and Nanotechnology at the University of Queensland, Australia. He then attended the Heart Research Center Göttingen (HRCG) at the University Medical Center, Göttingen, Germany, where he was awarded a postdoctoral fellowship from the German Cardiology Society for his work on developing protocols to generate human and non-human primate cardiac tissues. He moved to the School of Biomedical Sciences at the University of Queensland in 2013 after being awarded the prestigious NHMRC Peter Doherty Fellowship to continue his research using human cardiac tissues to study development and disease. He now co-heads the Cardiac Regeneration Laboratory with Dr Enzo Porrello.

adult cells, taken from the skin for example, iPSCs can be created. These iPSCs avoid the need for embryos and can be matched to individual patients. Indeed, together with collaborator Dr David Elliott, using this approach the team is able to generate cardiac tissue from specifi c patients or patient groups.

Combined with their 3D tissue engineering technique, the lab has created a powerful new approach. “This is extremely exciting as it means we can model human diseases in cultures for drug discovery, or use these cultures to devise personalised medicine strategies,” Hudson enthuses. However, producing 3D human heart tissues is a demanding feat, requiring millions of cardiac cells every week. By using a pioneering cardiac differentiation protocol, the lab is now able to produce an enormous 50 million cardiac cells every week, enabling the reliable fabrication of human cardiac tissues.

FACILITATING REGENERATION

Despite the astounding progress made, much remains to be done; a refl ection of the emerging nature of the concept. Although Hudson’s 3D human heart tissues are more mature than 2D cultured cardiomyocytes, they still do not exactly mirror the human heart, possessing a number of different characteristics. One example is the fact that the tissues are not terminally cell-cycle arrested (a hallmark of the adult heart). In other words, they still progress through the cell cycle.

In order to understand the molecular processes that drive terminal cell-cycle arrest and maturation in the adult heart, the lab is applying contrasting approaches: hypothesis-

driven investigation and the unbiased screening of compounds. In order to conduct such experiments, they have created a novel 96-well screening platform, able to measure – from each individual tissue – contractile parameters, such as force on contraction, and biological ones, such as proliferation.

This innovative platform is at the crux of the team’s efforts to facilitate the cheap and non-labour intensive production and analysis of tissues. In turn, this analysis is central to their efforts to fi nd the mechanisms that drive terminal cardiac differentiation in human heart cells, the process that inhibits regeneration. “Over the next fi ve years we hope to identify highly effective therapeutics to facilitate regeneration of the heart following injury,” Hudson reveals. Ultimately, they plan to use the knowledge gained to re-activate cardiac regeneration in patients and cure cardiovascular disease. “After identifi cation, we aim to progress to preclinical and subsequently clinical trials; however, this will take many years,” Hudson concludes.

A new cardiac differentiation protocol

Hudson’s method of transforming stem cells into cardiac cells has many advantages:

• Affordable and robust production of tens of millions of cardiac cells every week

• It can produce cardiac muscle cells and fi broblast support cells – both are needed to produce cardiac tissue that can generate force

• Optimised tissue engineering protocols ensure the production of force generating, serum-free cardiac tissue with enhanced maturity

INTELLIGENCE

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