Blood on demand
Within the next two decades, every hospital may be able to source blood for transfusions on demand, thanks to new techniques being developed in the University of Glasgow, using embryonic stem cells to generate red blood cells ....…
Add to this the ability to generate other human cells such as heart tissue cells, for use in testing drugs, plus blood vessel cells and even neural cells, to replace or regenerate damaged or diseased tissues, and the scope of the research seems even more remarkable. For example, a total of 24 million people in the EU and USA who suffer from ischemic limb conditions could benefit from therapies which help to regenerate tissues either by replacing damaged or lost cells with new cells or by “helping the resident cells grow for themselves.”
The idea of producing blood for use in transfusions was not Mountford’s original aim, certainly not on the industrial scale that is currently being envisaged – up to two million units a year, using industrial bio-reactors. As often happens in science and scientific careers, the path to Mountford’s current research project has taken a few twists and turns on the way – and under the surface there are several amazing surprises.
Mountford studied biological sciences at the University of Birmingham, where she also gained her PhD, specialising in blood development and working with progenitor cells from foetal livers and umbilical cords, before spending 12 months in Strasbourg studying molecular biology. She then spent two years in Oxford and returned to Birmingham again when she got a fellowship from the medical school, focusing on “intracellular signalling during haematopoietic differentiation.”
In 2000, in the wake of the Alder Hey scandal, which involved the unauthorised removal, retention and disposal of human tissue, including children's organs, and made it much harder to get access to human tissue for research, Mountford moved to Memphis, Tennessee, where she worked at St Jude Children's Research Hospital. Human embryonic stem cells had first been isolated from very-early-stage embryos in 1998 and three years later they were shown to generate blood – and this revolutionised Mountford’s ambitions for her research. In America at that time, she was able to experiment with adult stem cells, but US attitudes made it impossible to work with embryonic cells, and in 2002, Mountford headed back across the Atlantic to the faculty of medicine in Glasgow, to start research with human embryonic stem cells and continue her work on adult stem cells.
In Glasgow, thanks to funding from the Scottish National Blood Transfusion Service (SNBTS), Mountford was able to run her own research lab and that was when she started doing a collaborative project with her colleague Professor Tessa Holyoake, comparing aberrant leukaemia stem cells with normal stem cells to study how leukaemic stem cells resist death in response to targeted therapy. According to Mountford, blood stem cells are very difficult to obtain from normal individuals, so in search of a new source of these precious cells she started trying to make blood stem cells from embryonic stem cells (ESC), in the process making small amounts of red blood cells to demonstrate that these ESC-derived blood stem cells could become mature. “At that time, blood transfusion was not on the radar,” says Mountford. But then the American Department of Defense (DARPA) appeared on the scene, looking for a new source of red blood cells for transfusion on the battlefield. They set a challenge to scientists across the world to develop a process suitable for emergency use in the field – offering significant funding to advance the research. Mountford and colleagues from the Scottish, Irish and National Blood Transfusion Services applied for the programme but the theatre of war was soon supplanted by the operating theatre as the main thrust of Mountford's research.
Although it never did become reality, the “extraordinary” idea of a military blood factory evolved into a method for producing blood on demand for use in hospitals – what Mountford now describes as a “more realistic, normal idea”, with universal applications that would ensure a secure supply of blood for the future. Funding from the Wellcome Trust, SNBTS and, more recently, the Scottish Funding Council has allowed Mountford and her colleagues to advance this work, transferring laboratory protocols into clinically- usable processes and starting to look at the issues of scale.
This became the main theme in her research from 2008 onwards, with other applications such as blood vessel cells (for regeneration) and heart tissue cells (for testing the safety of new drugs) also in development.
“Manufacturing blood for transfusions is only one application,” says Mountford. “By generating heart or liver cells, for example, researchers could avoid late-stage failures with drug trials and make drug development faster and safer.” Theoretically, stem cells could also be used to develop regenerative medicines for diverse applications – including everything from solving the problem of hair loss to the replacement of neural cells in degenerative conditions such as Parkinson’s. The level of complexity of some of these applications is much higher than for blood transfusion, says Mountford. For example, neural cells would have to integrate into the brain and re-establish the correct connections, whereas blood is administered directly into a vein and functions as a cell suspension rather than as complex structured tissue.
Red blood cells do not have a nucleus so have fewer safety concerns compared to muscle, nerve and liver cells generated from embryonic stem cells, for example. But if scientists can crack this first basic step, the possibilities are endless, with cost a major part of the equation. According to Mountford, every unit of blood for transfusions costs at least £140 to supply (even more with add-ons such as testing and public awareness campaigns) – and blood services account for a massive three per cent of the total NHS budget.
The biggest technical hurdle, says Mountford, is getting the nucleus out of the cell, “but we are making progress,” she adds. This is what happens in nature, she explains, so the cells can carry oxygen more efficiently and travel through the narrow capillaries of the blood vessels. In addition to the fundamental science involved, the other major hurdle is to upscale production to industrial levels. One unit of blood contains about two trillion red blood cells and the NHS uses about two million units a year – “astronomical” amounts compared to current laboratory levels. The body is very efficient, says Mountford, and the bone marrow produces two million new red blood cells every second. So far, Mountford and her team have managed to produce a billion cells (109) or roughly a teaspoon from a single experiment. The number needed for trials is about 1015 cells, which would make about 500 – 1,000 units of red cells, while commercial-scale production would be at least 1,000 times greater.
“The other sticking point is numbers,” Mountford explains. “Unlike the cells which develop in humans, cells which grow in vitro like low densities, about one million per millilitre. At that density the generation of a single unit of red blood cells would take 1,000 litres of growth medium at a cost of about £300 per litre.” This is not economically viable, says Mountford, so new culture conditions will be needed that enable the cells to grow and mature at much higher density. The team in Glasgow is now working with colleagues at Heriot-Watt University to address these bio-processing challenges.
As well as quantity, the researchers also have to think about quality issues, including the use of animal products and the possibility of contamination during the manufacturing process. Foetal calf serum is used in the process to grow human cells simply because it is available in bulk, but there is a possibility of transmitting animal diseases or that some of the animal proteins may attach to the surface of human cells, risking cross-species immune responses if the cells were put into people. “In the early stages of the project, animal products were used, but we are eliminating these and replacing them with clinical-grade human or recombinant reagents to eliminate these risks,” Mountford explained.
Another issue is the need to harvest cells – or separate the mature cells from immature cells. The engineering challenge this presents is not unlike a sewage works, says Mountford, potentially including very similar fluid dynamics and similar decisions – i.e. “How do we separate lighter erythrocytes from denser nucleated cells?” and “What is better: individual batch production or continuous flow systems?”
Typically, red blood cells live for 120 days in the body, so when a unit of blood is donated, cells of varying ages will be collected. However, in vitro generation should produce a more uniform product to maximise efficiency at the same time as achieving mass production. Blood donations can be stored for about 35 days, but scientists still argue about whether blood more than three weeks old is less effective than fresh blood. Mountford adds:
“We don't know how long our blood will last, but our major advantage is that we will be able to supply on demand, evening out peaks and troughs of current supplies and theoretically supplying the NHS with universal donor (O-) red blood cells that have been freshly grown and distributed.”
The stem cells themselves must meet the highest of quality standards, primarily the Good Manufacturing Practice (GMP) standard, and the project sources clinical-grade cells from Roslin Cells near Edinburgh, one of the only suppliers in Europe which produces “true” GMP cells. Mountford says: “We develop processes in the lab that will drive the embryonic stem cells to differentiate into red blood cells, but these processes need to be reviewed and amended to comply with GMP standards and to produce clinically- acceptable cells. This is a very different level of rigour that is not familiar to most academic labs, but Roslin Cells and the SNBTS have invaluable expertise in this area and are central to the overall project.”
The newly-extended project brings together a multi-disciplinary team of biologists and engineers, and one of
the most curious aspects is the role of social scientists. The use of embryonic stem cells is both novel and controversial, says Mountford, so it’s vital to “manage public expectation” and consider the impact on society at large, especially in view of previous issues such as the Alder Hey “scandal” and fears generated by variant CJD and HIV Aids. People need to be reassured that the new kind of blood is perfectly safe as well as “ethical,” but equally important is the need to ensure that donors continue to give blood and don’t get carried away with the idea that blood will soon be easy to produce on demand – a prospect still more than a decade away.
“In seven years,” says Mountford, “we will know whether it works or not. Clinical trials could begin in five to six years and it would be 10 years before it becomes widely used.”