NIRM

Help the body to repair itself

Amsterdam 2016

Help the body to repair itselfNIRM

NIRM NIRM

Introduction

Presented here is a book describing the ground-

breaking results that have been achieved within

the NIRM LSH-FES consortium. LSH stands for ‘Life

Sciences & Health’, or rather the application of life

sciences to the benefit of our health, and FES for the

‘Fund for Economic Structure Enhancement’, finan-

ced by the so-called natural gas profits. NIRM started

its activities in 2010 upon the Dutch government’s

approval of a joint proposal, which was submitted

by a combination of many partners. These included

small and large businesses, multinationals, academic

and medical research groups, patient organisations,

and the ministries of Health, Welfare & Sport, Edu-

cation, Culture & Science, and Economic Affairs.

The FES grant was used to establish a public-private

partnership: every single euro invested by the part-

ners was matched by the government. After six years

of hard work, the results can be directly applied in

the treatment of patients or used for research that

is closely linked to patients.

The consortium has thus proven that it has used the

research funds to contribute to the improvement

of our national public health and boost economic

activity in the Netherlands. A vast number of these

results are presented in this book. The most recent

developments in this exciting field will astonish you

time and time again. I therefore trust that you will

greatly enjoy reading this book!

Herman Verheij

L S H - F E S S E C T O R C O O R D I N A T O R

Regenerative medicine is in the spotlight. In-depth

studies into stem cells, biomaterials and networks of

signals within and outside cells have taught us that

there is still plenty of room for improvement in the

techniques that are currently used to repair tissues

and organs. New, powerful techniques have been

developed – take, for instance, the printing of bio-

materials, or the possibility of turning a common

blood cell into a stem cell again and then having it

differentiate into almost any cell type we want. Just

a decade ago we would hardly have thought that this

was possible. However, this is not science fiction;

this is fact. True, we are still mostly at the lab level,

but this is where all the innovations towards better

treatments begin – innovations that also boost our

knowledge economy. Patients with diabetes or old

age blindness will probably be able to benefit from

these new developments in the near future. The

foundations have been laid; it is now all about the

next step. We are not like salamanders. When we

lose a leg, it will not spontaneously grow back again.

Yet new knowledge on full bodily repair might make

it happen one day. Would you call this science fic-

tion? Or is it fiction that may become fact? NIRM is

proud of the part it plays, however great or small, in

turning dreams into knowledge.

Ruud Bank & Elaine Dzierzak

P R O G R A M M E D I R E C T O R S , N E T H E R L A N D S I N S T I T U T E O F R E G E N E R A T I V E M E D I C I N E

ContentIntroduction 3

When stem cell research and tissue engineering meet 4

Horses for courses – printed cartilage for joint repair 6

Design your own heart valve 9

A shove in the direction of bone or cartilage 12

Build a tailored test kit 18

Stem cells surviving on synthetic hydrogel 20

Reparation with stem cells 22

Never eating toast again 24

Make your own research material 27

The heart still has some secrets to reveal 28

Repairing liver damage with the patient’s own cells 32

Barcoded blood cells 35

Are the regulations ready for regenerative medicine? 36

NIRM partners 38

Colophon 41

3

NIRM Infographic

story: René Rector image: Parkers

When stem cell research and tissue engineering meet

Building entire organs or tissue structures outside the body and using

them to help the body repair itself is what tissue engineering is all about.

However, this is trickier than it sounds, because how do you make sure

that only the part of the tissue you need grows?

1 Doctor extracts stem cells from the patient’s body.

2 A scaffold is built in the lab. This can be done in sever-al ways. It can be printed using a 3D printer, or the biodegrad-able material can be moulded into the right shape.

NIRM

Sometimes, tissues and organs are

damaged to the extent that they

can no longer repair themselves.

When the tissue or organ is essen-

tial for a reasonable quality of life,

patients usually have to rely on

treatment of their symptoms or

a transplant. However, donor

material is on short supply.

Furthermore, after transplanta-

tion patients have to take medica-

tion for the rest of their lives to

prevent rejection. Regenerative

medicine works differently.

Instead of relying on a donor,

autologous cell material is

cultivated outside the body

and then implanted back into

the patient.

This is a complicated process. Not

only does the treating physician

have to be able to cultivate suffi-

cient tissue from the patient’s

cells outside their body, this tis-

sue also has to develop in exactly

the right way so that it will func-

tion as intended. As an aid, tissue

engineers often start off by mak-

ing a scaffold from biodegradable

material outside the body. By

making a scaffold from “smart”

material and then injecting

autologous cells onto it, tissue

can be grown in the lab to have

the right shape and function.

“Smart” means that instructions

are built into the material so

that the cells will know what to

do. Stem cells work best for this

job because they still have to

develop.

3 Stem cells are injected into the scaffold. The scaffold “directs” the stem cells to develop in a specific way.

4 The new construct is surgically implanted in the right spot. The body then takes over the repair work. Because it has been cultivated from the patient's own cells, the implant will not be rejected.

54

NIRM Interview

Why is research on horse cartilage so important?“Most domesticated animals are important for

their products. Horses, however, are important

for their movement. They have been used by

armies, in agriculture and for transport, and today

are used primarily in sports. Their locomotor sys-

tem is essential. What I do is comparable to sports

medicine for horses. Most of the health problems

we come across in horses affect their musculo-

skeletal system, with damaged cartilage in the

joints being one of the main causes of poor

performance.”

Can’t such damage simply be repaired?“Cartilage is complicated stuff. Unlike almost all

other types of tissue, in adults it doesn’t repair itself

after an injury. Cartilage cells need an extremely

long time to produce new collagen: it would take

around three hundred years for joint cartilage to

completely regrow. We’ve known that cartilage is

tough to repair since scientists first wrote about it

in 1743, but we have yet to find a solution. Cur-

rently, a hole in someone's cartilage is usually

repaired by drilling small openings in the underly-

ing bone so that bone marrow cells can fill the hole

with new tissue. However, the quality of the result-

ing scar tissue is much worse than the original car-

tilage. Another option is to replace the entire joint

with an artificial one, but artificial joints are not

ideal and have a limited lifespan. We are now look-

ing for a solution using stem cells so that cartilage

can finally repair itself.”

How would that work?“Though cartilage cannot be reproduced, you can

implant a biomaterial (called a scaffold) in which

cartilage-producing cells can survive. We are col-

laborating with the University Medical Center

Utrecht (UMCU), where they are working on a

technique to produce such biomaterial using 3D

printers. There are still quite a few variables in

their research. For example, you could use carti-

lage cells, cartilage stem cells or more general IPS

cells. The researchers are in NIRM also working on

a biomaterial with ‘vesicles’. These are pouches

filled with substances such as RNA that regulate

Veterinarian and Utrecht University Professor René van Weeren’s research

focuses on bioprinting cartilage for horses. Damage to cartilage is something

equestrians would love to be able to remedy. But his work offers prospects for

treating cartilage injuries in human patients too, says Van Weeren.

“ It takes around three hundred years for joint cartilage to completely regrow.”

Horses for courses – printed cartilage

story: Rineke Voogt

NIRM

for joint repair

A horse is placed on a special treadmill after treatment. Using cameras and measurement equipment, researchers monitor the exact joint pressure and whether it is the same in all four legs.

Horses for courses – printed cartilage

76

NIRM Horses for courses – printed cartilage for joint repair

communication between cells. If these vesicles

can prompt your own cartilage cells to produce

collagen again, you don’t even need stem cells

anymore.”

IPS cellsInduced pluripotent stem cells (IPSCs or IPS cells)

are stem cells made from adult skin cells. These

stem cells can develop into a wide range of

specialised cell types. The advantage of using

these cells is that they can be harvested from

patients themselves, thus preventing problems

with the immune system.

Have these techniques been applied in practice yet?“You can test cell growth in a biomaterial in vitro,

but we’re conducting the tests in horses. Fortu-

nately, it is far easier to take various measurements in

horses than in classic lab animals like mice or rats.

For our tests, we inflict a small injury to the horse’s

cartilage and then administer the intervention with

the printed biomaterial. We thoroughly examine the

horse before, during and after the experiment. We

place the horse on a treadmill, for instance, to mea-

sure the exact force exerted on the ground, the angle

of the joints and which leg has gone lame. We also

analyse all its movements using cameras, and a kind

of keyhole operation even lets us visually monitor if

and how the cartilage is repairing itself. Biomarkers

in the joint fluid can tell us if any inflammation has

occurred and whether any additional collagen has

been produced or broken down.”

And?“We still have a long way to go – generating proper

cartilage tissue would be a feat worthy of the Nobel

Prize. To begin with, the material is still not ideal.

In some tests, the cartilage quickly broke down

again. The procedure itself also has to be improved.

The downside of using horses is that the implanted

material is put to the test straight away, since the

animal puts pressure on its leg immediately after

the operation, when it gets on its feet. In this

respect, working with humans is easier. You can

simply tell them to stay off their feet for a while.”

Painless wear and tearDamage to joint cartilage is not noticeable at first as

there are no nerves in cartilage tissue. But, once inju-

red, the rest of the cartilage also wears out faster. This

is when it becomes painful, because the underlying

bone – which does have a lot of nerves and is there-

fore very sensitive – is eventually exposed.

This research also opens up prospects for people with joint problems. How big is the leap from horse to human?“At the start of our study we examined the thick-

ness, composition and other characteristics of car-

tilage in various animals. We compared some 120

animals across 58 mammal species – all of them zoo

animals sent here for analysis after they died. We

found that the cartilage in many animals that weigh

over one kilogram is very similar. In horses and

humans it’s even extremely similar. Therefore, if we

manage to repair it in horses, it’s almost a foregone

conclusion that we can do it in humans. That’s also

why we are able to work so closely with researchers

on the human side of things at the UMCU: the tar-

get group for this research is not only horses, but

humans as well.”

“ Generating proper cartilage tissue would be a feat worthy of the Nobel Prize.”

NIRM Case study

There is an urgent need for implantable heart valves

that more closely resemble the real thing. The artifi-

cial valves currently in use have a lot of drawbacks.

Carlijn Bouten, Professor of Biomedical Engineering

at Eindhoven University of Technology, explains, “A

mechanical prosthesis is not alive. It is made of syn-

thetic material and metal, and you can hear it tick.

Even worse, they knock blood cells to pieces. This

means you have to take medication to prevent

thrombosis for the rest of your life. But these blood

thinners also make bleeding difficult to control. In

other words, though the patient’s quality of life

improves, the cost is another disorder.” A biological

valve from a donor (human or animal) is also far

from ideal, because it has to be replaced after fifteen

years. This means that young patients – every year

six thousand European children need a new heart

valve – face a series of risky operations because arti-

ficial heart valves cannot be tailor-made. Moreover,

donor valves are scarce.

Design your own heart valve

People with a leaking or constricted heart valve can have an artificial replacement

implanted. But there are drawbacks. A mechanical artificial heart valve requires

lifelong use of medication, and a valve prosthesis made of animal material wears

out quickly. Moreover, neither type of valve grows with a patient. Carlijn Bouten is

working on an alternative: an implant that attracts autologous cells as soon as it

is implanted and uses them to build a new heart valve.

1 The heart pumps blood around the body continuously.

2 A malfunctioning heart valve makes the heart work too hard. Ultimately, this leads to complications.

3 The tissue-engineered artificial heart valve is implanted to replace the patient’s own defective heart valve.

story: Rineke Voogt images: Hartstichting

98

NIRM

Bouten and her colleagues believed there had to

be an alternative. They had already worked on a

new type of tissue-engineered artificial heart valve

that they cultivated in the lab. “Heart valves open

and close a hundred thousand times a day. We

know that live valves perform much better. They

do not wear out and are self-healing. They also

adapt to new circumstances, such as increased

blood pressure. Tissue engineering is already being

used to create live prostheses, and we want to do

the same for heart valves.”

LifelongA tissue-engineered valve made of autologous

cells would have none of the drawbacks of standard

artificial heart valves – no risk of thrombosis or

rejection symptoms. Furthermore, live tissue-

engineered valves can grow with patients and

last a lifetime.

However, a lab-grown heart valve has drawbacks

of its own. For example, it is not clear who actually

owns the valve – the researchers, the patient, the

doctor? There is also a high risk of infection. More-

over, cultivation takes more than eight weeks and

is very expensive. The Eindhoven researchers have

therefore opted to take a different approach: the

implantation of a lifeless framework – known as a

“scaffold” – which develops on its own into a fully

functioning heart valve inside the body.

Auxiliary substances The principle is simple: take a solid but porous

plastic-like material that is degradable in the body,

implant it, and allow the body to populate the

scaffold with its own cells. According to Bouten,

“The body is great at making its own tissue. Take

the way it repairs wounds. Cells are attracted to sites

of inflammation. The only thing you need to do is

steer the process in the right direction.” Bouten’s

group directs this growth process, for example, by

injecting certain active molecules into the scaffold

that promote healing by attracting monocytes

(a type of white blood cell) in the blood. Inside the

scaffold, the cells gradually form a strong tissue.

The Eindhoven scientists have already successfully

used this method to replace blood vessels.

The scaffoldThe implant – the scaffold that forms the basis for the

heart valve – consists of wires of biologically degradable

polymers a thousandth of a millimetre in diameter.

These scaffolds are built by Xeltis, a biotechnology com-

pany. “It’s like making candy floss”, Martijn Cox at Xeltis

explains. Under an electric current, a long, thin thread is

spun around a mould for a heart valve to make a 3D

network that autologous cells can latch onto.

Special groupsThe mechanics of the valve seem to work well. The

challenge now is to have the valve grow precisely

the right tissue within the body. A heart valve has

to be strong, but also flexible, and it has to have

the right shape. Moreover, the scaffold has to break

down at a certain pace, neither too slowly nor too

quickly. “We still have to fine-tune this process”,

Bouten says. That work, however, takes place out-

side the body, at the Eindhoven lab. The researchers

place the scaffolds in a bioreactor where they are

supplied with an artificial blood flow to reproduce

conditions in the body as closely as possible. A

“human model system”, they call it. “We still have a

lot of testing to do”, continues Bouten. “We want to

find out how quickly the tissue grows, and how the

breakdown of the scaffold material can be switched

on and off. A scaffold has certain characteristics,

but they change as the tissue grows. Compare it to

passing the IKEA test: you first have to make sure

the valve can ultimately do what it’s meant to do

and will carry on doing it.”

NIRM

4 The artificial valve is made of biodegradable material.

5 A close-up of the material (a type of plastic) shows that all kinds of substances can be attached to the scaffold.

6 The scaffold is supplied with molecules that attract monocytes – a type of white blood cell – to nestle within.

7 Attracted by the bait substances, white blood cells colonise the scaffold.

8 Due to the presence of the white blood cells, the scaffold is gradually reinforced with collagen.

9 While the scaffold is broken down, its task is taken over by autologous tissue.

10 The body has replaced the old, defective valve with new material.

Design your own heart valve

In the “in vivo situation” mimicked at Bouten’s

lab, she can also simulate the conditions of a sick

or old body. Because wounds heal more slowly in

diabetics, for example, than in healthy people, this

would have to be taken in to account when building

heart valves for diabetic patients. Similarly, cell

characteristics differ between old people and

young people.

The scaffold method offers solutions for these

more difficult conditions too, by means of adding

auxiliary substances. Bouten and her colleagues

want to find out which substances work for several

key patient groups. “Naturally, the heart valve can

be injured again, so we have to determine whether

this technique is also suitable for diabetics, for

instance.”

SheepThe next stop for this treatment is lab animal

research. Although the human model system is

actually more complicated than lab animal testing

in some ways – after all, you can use it to simulate

a sick patient – the technique has to be proven

effective in lab animals before it can be used on

humans. The researchers have already selected

the ideal candidate: sheep, which were previously

used to test the lab-grown valves. Sheep also

represent the worst case scenario because of the

pace at which their heart valves can calcify. If the

researchers manage to grow a functioning, healthy

heart valve in sheep, it is very likely that it can also

be done in humans.

That final stage – implanting scaffolds in people

with poorly functioning heart valves – is still

some way off. However, once the valve has proved

effective in sheep for a full two years, the road to

the first clinical trial is open.

1110

NIRM Interview

story Elles Lalieu images UMC Utrecht

A shove in the direction of bone or cartilage

NIRM

Stem cells are cells that can still

become anything they like. But how

do these cells decide what they will

be in the end? We are getting better

and better at answering that ques-

tion. Different groups within NIRM

are trying to make stem cells develop

into bone or cartilage tissue – and

they are well on their way.

A shove in the direction of bone or cartilage

1312

NIRM A shove in the direction of bone or cartilage

face and the cell’s behaviour, Van Blitterswijk and

his team developed the Topochip, a chip with over

two thousand different surface structures.

Stem cells are seeded onto the chip and subse-

quently attach to one of the available structures.

Cells seem to “read” the structure on which they

grow, just like the blind read braille. Stem cells spe-

cialise in a certain direction, depending on the sur-

face structure. On one surface they evolve into

muscle cells, for instance, whereas they grow into

bone cells on the other.

Building blocksThe structures on the chip are combinations of

three basic forms: a circle, a rectangle and a trian-

gle. The Topochip comprises over 2,000 structures,

but a total of 158 million combinations are possible.

All these combinations are stored in a library. For

example, if researchers want stem cells to turn into

bone cells and know that the circular structures are

the most suitable for that purpose, they can make a

new chip with all kinds of related circular struc-

tures to find out which surface is the best stimulant

for the desired development.

While the Topochip focused on individual cells, Van

Blitterswijk has meanwhile also developed a system

to grow mini organs on different structures. “We

use quite a few 96-well plates in the lab”, he

explains. “These allow us to test 96 conditions in

one go. What we have done is cover the bottom of

each of these wells with 580 tiny micro cultivation

containers – so that we can now grow 50,000

micro-organs on one plate.”

On top of the micro cultivation containers is a

layer of gel where the stem cells are seeded. Van

Blitterswijk: “The cells cannot attach to the gel,

so they sink through the gel into the containers.

That is where the cells coagulate and eventually

form a small ball of approximately one tenth of a

millimetre. We can harvest these balls and bring

them together into a greater mould, thus ending

up with a piece of a puzzle of approximately one

millimetre. Such pieces can be used as building

blocks towards tissue replacement.”

The collection of different types of cells in one con-

tainer makes it possible to create complex tissues,

such as a bit of bone or cartilage with blood vessels.

The bioprinter in the biofabrication facility. The printer is not only used for orthopaedic research, but can be widely applied.

NIRM

Cell biologist Clemens van Blitterswijk focused his

doctoral studies in the 1980s on the development of

ceramic ossicles. He is now professor of Regenera-

tive Medicine at Maastricht University. Over the

past thirty years he has conducted a great deal of

research in the area of tissue regeneration: creating

natural tissue for the purpose of replacing defective

body parts.

“My research group has now created the smallest

ossicle, the stapes, from cartilage”, Van Blitterswijk

says enthusiastically. “In the ear this ossicle is made

of bone; unfortunately, that is still a step too far.

But I do see it as a proof of concept. We can easily

make a great number of ossicles, at very low cost.

This is key, since affordability is one of the chal-

lenges for regenerative medicine: we can make all

sorts of things, but if the costs are prohibitive, it is

no use to anyone.”

TopochipThe stapes made of cartilage is a practical example,

and a result of extensive research. Stem cells grow

on different surfaces, but not in the same way on

each and every surface. The form and structure of

the surface determine the stem cell’s behaviour as

well as the type of cell it eventually grows into. In

order to examine the relationship between the sur-

Previous pages: A small part of printed material in a petri dish.

Cartilage cells in a micro cultivation container. The cells coagulate and eventually form the building blocks for micro-organs.

1514

NIRM

The NIRM researchers implanted one such culti-

vated building block in mice, where it shaped into

bone in a spot where normally no bone is found.

Fleece sweaterPrinting entire tissue with living cell blocks is a step

further. Bioprocess technologist Jos Malda saw an

opportunity in bioprinting a few years ago, and he

jumped on it – with success. The University Medical

Center Utrecht is now the proud owner of a special

biofabrication facility, which is entirely focused on

3D printing.

What makes the bioprinter so suitable for repairing

cartilage defects? Natural cartilage has a layered

structure which is ideal for reproduction using a

3D printer. The “ink” that Malda uses is a hydrogel

filled with stem cells, cartilage-forming cells or

a combination of both, plus growth factors.

As the cells in the gel have to stay alive during

and after the printing process, the hydrogel has

been made to provide them with an ideal living

environment. “The hydrogel is a weak substance, a

bit like gelatine”, Malda explains. “It is, of course,

not something you can walk on. That is why, as

part of the NIRM project, we have tried to do clever

things with it, so that the material gained more

strength. We first tried to print the gel on a mesh

of thick fibres, like fortified concrete.” Although

the result was certainly solid, it needed relatively

extensive fortification making the implant rather

inflexible. The fibres had to be thinner. Malda:

“We managed to make thin fibres, comparable to

the fibres of a fleece sweater. These fibres are in

themselves not robust, but this changes when you

print them together with the hydrogel. The forces

exerted on these fibres keep the entire construct

together. One plus one does not equal two in this

case, but fifty.”

Unravelling mechanismsMalda is now making implants with a combination

of thick and thin fibres. The thick fibres are more

suitable for creating bone and the thin ones for

cartilage. Printing the tissue in combination with

supportive materials has also enabled him to

make different shapes, such as slices or tubes. The

progress that he made soon allowed the printed

The bioprinter in action. The material is deposited in layers.

NIRM

implants to undergo their first test. Malda and his

team have tried to replace the entire shoulder of

a rabbit by printed tissue. Whether it worked,

remains the exciting question for now: the results

will be evaluated over the next few months. There

is funding for this research for the next five years,

so the story of these experiments with joint replac-

ing tissues will definitely not end here.

While improving their ability to create different

tissues, do Malda and his group now also under-

stand exactly why a stem cell becomes a muscle

cell in one instance and a bone cell in the next?

Malda: “Our insight is increasing, but we haven’t

yet reached the stage where a stem cell forms

bone whenever we like it to.” According to Van

Blitterswijk we are only now starting to understand

how it works. “We can now see how cells respond.

If we grow organoids in the shape of a triangle, for

instance, we see high concentrations of blood

vessels in the vertices. Why? Because of the high

concentration of the VEGF growth factor there,

which is needed for the formation of blood vessels.

But what causes the high concentration of growth

factor in these exact spots? We hope to unravel

these types of mechanisms in the next five years.

We will not be able to truly predict a stem cell’s

behaviour until then.”

A shove in the direction of bone or cartilage

In Van Blitterswijk’s lab, the stirrup bone or stapes, one of the ossi-cles, was made out of cartilage. Normally, the stapes is made of bone, so this cultivated speci-men is not functional, but it does show that it is relatively simple to create a great many ossicles.

1716

NIRM

Sometimes, it is not clear how medicines will

affect patients. The effects of a medication to treat

intestinal cancer, for instance, depends on the

genetic characteristics of the tumour, which can-

not be seen from the outside. What’s needed, reali-

sed Hans Clevers and his colleagues at the Hubrecht

Institute (part of the KNAW Netherlands Academy

of Sciences), is to be able to test the medication on

the patient’s cells first, without the patient suffe-

ring any consequences. That way, you can better

predict the response. If the test shows that aggres-

sive chemotherapy has no chance of success, you

can spare the patient a pointless treatment.

Patient test kits can be built by taking a small

amount of tissue from the patient’s body, cultiva-

ting it into miniature organs and then testing the

medication on them. Researchers at Clevers’ lab

figured out a way to do this: using growth factors,

the patient’s stem cells can be stimulated to

multiply and specialise (see also Repairing liver

damage using patient cells). The result is a so-

called organoid that is not only similar in shape

and function, but also has the same genetic code

as the original organ.

“You can then expose these petri dish organoids

to medications to see what the effect will be”,

Rob Vries explains. He is the managing director

of the HUB Foundation, founded by the Hubrecht

Institute to test medications on a large scale

and gain a better understanding of why patients

respond to them so differently. Their work is still

in the research phase. When a doctor prescribes a

treatment for a patient, Vries and his colleagues try

to make a prediction on the basis of the patient’s

cell material. “If we manage to consistently make

accurate predictions, we will be able to make real

recommendations one day.”

Testing medicines on organoids has also turned

out to be useful for disorders like cystic fibrosis.

Hubrecht researchers have even managed to cure

the cultivated mini bowels of a cystic fibrosis

patient by replacing a defective gene with a healthy

one. This offers hope for a potential treatment for

the disease.

Testing existing medications on cultivated patient

cells allows for tailor-made medications. Taking

this a step further, brand-new medicines could

also be tested in petri dishes. This is exactly what

the biotechnology company Pluriomics does,

focusing on the heart. Cardiac muscle cells, culti-

vated from what are known as induced pluripotent

stem cells, make it possible to study the effect of

a medication on the heart. The cells behave almost

identically to how they do inside the body.

“You can see them contract, or ‘beat’”, says

Marijn Vlaming, a researcher at Pluriomics.

“Using a type of ECG test, we can then see if a

substance causes arrhythmia, for instance.”

The test organoids have various applications.

They are particularly useful for the development

of medications, says Vlaming. “Many medicines,

cancer drugs for instance, pose a risk to the heart.

Currently, their safety is often tested in animal

models, but something that is dangerous to a dog’s

heart is not necessarily dangerous to a human heart

too, or vice versa. Our test seeks to provide greater

certainty. This technology is already replacing lab

animal testing, but it will still be a while before

lab animals become completely unnecessary.”

With mini test organs offering so many uses,

Pluriomics is currently developing a “toolkit”

with which researchers can perform their own

tests on cardiac muscle cells they cultivate in their

labs. “This is convenient”, Vlaming says, “because

academics often want to do things themselves.

Plus, the more the method is used, the sooner and

more widely the technology will be accepted.”

NIRM Case study

How effective medicines are in treating

specific disorders differs considerably

from one patient to another. By ex-

tracting and cultivating stem cells from

a patient’s body, the effectiveness or

risk of medications can be tested

beforehand – making it possible to

identify the most effective medication

for that patient.

Build a tailored test kit

Cardiac muscle cells grown from human stem cells shown in a “multi-electrode array” dish. Electrodes are used to measure the effect of medication on the contraction of the cardiac muscle cells.

Microscopic image of the structure of cardiac muscle cells grown from human stem cells. This sarcomere structure, which helps cardiac muscle cells contract, consists of the overlapping proteins myosin (red) and actin (green).

Contracting heart muscle cells grown from human stem cells in a petri dish.

story: Rineke Voogt images: Marijn Vlaming

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Stem cells surviving on synthetic hydrogelIt looks like a landscape in a science fi ction movie, but this is an image of a natural extracellular matrix. Chemist Patricia Dankers tried to syntheti-cally copy this matrix in order to serve as a syn-thetic base for the growth of stem cells. Stem cells in the lab now grow on a natural hydrogel called matrigel. It may never be clinically applied, how-ever, since tumour issue is requiered fot the preparation of the matrigel. Dankers: “Matrigel contains around ten thousand different compo-nents. The trick is to identify the relevant ones and reproduce them synthetically. Five years ago, we thought that it would be a straightforward job, but nothing could be further from the truth.”

The idea was to use small building blocks to create a larger structure, held together by hydro-gen bridges. Dankers and the research team devel-oped building blocks based on the ureidopyrimidi-none (UPy) group. If you couple other molecules to these building blocks, you end up with different UPy-blocks with widely varying functions. It is the combination of specifi c building blocks that will get you a specifi c bioactive hydrogel.

Over the past few years, the researchers have cre-ated a structure to which three or four UPy-blocks can be added. Dankers: “The stem cells survive and that in itself is quite an achievement, as there have been many synthetic gels made over the years in which they simply died. However, it will take years before we create a synthetic gel that also provides us full control over stem cell behaviour.”

story: Elles Lalieu

Report NIRM ?

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Testmedium A

Testmedium B

NIRM Infographic

Reparation with stem cellsThe cultivation of body material outside the body so that you can tinker

with it appeals to the imagination. However, you can do far more with

stem cells. You do not always need to create entire grafts and even

without placing them back, stem cells are of great value to patients.

story: René Rector image: Parkers

1 A doctor extracts stem cells from the body.

2 The stem cells are cultivated outside the body, until their number suffi ces.

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Testmedium A

Testmedium B

3a After differentiation, the healthy cells are injected into the patient again, where they can multiply further. The great advantage: the cells are autologous, so the patient will not suffer any rejection symptoms.

3b The cultivated tissue serves as a test medium in order to see how the patient responds to certain medication. This is useful when patients differ strongly in their respon-ses to the specifi c medicine or when there is a chance of it being harmful.

If an organ is defective, you can

try to recreate the entire organ

in a cultivation fl ask or with a

printer. Th is is not always neces-

sary however for each and every

tissue. Sometimes it will suffi ce to

grow something new at the place

of the defect. How do you do this?

Th is type of research basically

starts out in the same way as tissue

engineers do, by extracting stem

cells from somewhere in the body.

Th e researchers then cultivate

those stem cells outside the body,

after which they are diff erentiated

into the right kind of cells. Th ese

cells are subsequently injected

into the body again, where they

can take on the task that the body

itself is no longer able to perform.

Th is sounds simpler than it is. In

reality, each step in the process is

a challenge. First of all, there is the

diffi culty of fi nding the right stem

cells. Next, they have to be grown

in a petri dish. Probably the most

complicated challenge is to make

certain that it is safe to return

them into the body.

Th e latter is not always necessary.

For various disorders drugs are

already available, but they pro-

voke widely diff erent responses

in patients.

One cardiac or cancer patient

may greatly benefi t from a certain

drug, whereas in another it may

be totally ineff ective or actually

harmful. In such cases, what

doctor would not like to be able

to predict whether their prescrip-

tions are indeed going to help?

We are improving our ability to

do just that, as researchers are

now able to cultivate patients’

autologous tissue outside the body

with the help of stem cells. Th e

cultivated material then serves as a

“guinea pig” to predict the eff ects

of the medication.

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Life without properly functioning salivary glands is pretty uncom-

fortable, and repairing them has turned out to be difficult. At the

University Medical Center Groningen, Rob Coppes is working on a

solution: the cultivation of salivary gland stem cells.

Constantly having a dry mouth, not being able to

talk or swallow properly, damaged teeth, sleep

deprivation: not having enough saliva is the source

of some unpleasant complaints, which are a daily

reality for people who have to undergo radiation

treatment due to a tumour in their throat or head.

The radiation that is meant to combat the tumour

damages their healthy salivary glands. These glands,

which can be found under the ear and tongue,

among other places, are highly sensitive to radiation.

“It is a nasty side effect of radiation”, Rob Coppes,

Professor of Radiotherapy at the UMCG explains.

Saliva is essential to oral health as well as speaking

and swallowing. “Try eating two pieces of dry toast

one after another – that is how it feels if you have

to eat without saliva.”

Approximately forty per cent of the people who

undergo radiation therapy in the head-and-neck

area experience problems with their salivary glands,

Coppes estimates. That boils down to some four

hundred patients each year. They end up with xero-

stomia: dry mouth syndrome. Marijke Baks is one of

them: ever since she underwent radiation treatment

in 2007, her saliva production has virtually stopped.

“It is something that you really need to learn to live

with: for instance, you have to walk with your

mouth closed, because otherwise it gets too dry.

Singing is no longer possible, and it is especially

difficult at night, as you often unknowingly sleep

with your mouth open.” And that is not the end of

it. Sports are out of the question without artificial

saliva, intimacy is more problematic, and hot

weather is also a bother: drinking too much water

actually gives you a dry mouth. “You have to adapt

the way you live”, Baks says. “Meals are difficult;

they always have to be moist. I often eat mashed

potatoes and vegetables, and whenever I eat a

sandwich, I have to take a sip with each bite.”

Although there are makeshift solutions available

– artificial saliva, or always having a bottle of water

Never eating toast again

story: Rineke Voogt image: René den Engelsman

Interview NIRM

at hand – there is still no remedy for people with dry

mouth syndrome. Coppes and his team are looking

for a way to repair the salivary glands using stem cell

therapy. “The method is actually similar to a bone

marrow transplant”, Coppes explains. “We take

some of the patient’s healthy cells, cultivate them,

and then place them back, returning healthy mate-

rial that came from the patient’s own body.”

The stem cells can be directly extracted from the

patient’s salivary glands, which are still intact prior

to the radiation treatment. A small cut under the ear

suffices for a biopsy. Coppes: “Of course, you cannot

take away the entire gland – there is a limit to the

number of cells that you can harvest. But we have

found a way to grow six thousand stem cells outside

the body from a single stem cell within seven

weeks.”

There was no doubt about the possibility of cultivat-

ing salivary gland stem cells in itself. “After all, if

you chew chewing gum for two weeks, your salivary

glands grow as well.” But it proved to be more diffi-

cult to mimic this process in the lab. The question

was: what made a stem cell behave like a stem cell?

And how could the researchers get them to multiply?

Those were the tough nuts that Coppes’ team had to

crack. Growth factors (chemical substances natu-

rally present in the body that promote cell growth)

proved to play an important role, but finding the

right growth factors was far from easy.

The research conducted by Coppes’ team is the first

investigation into salivary gland stem cells. There is

one significant difference with research in other

organs: other organs are often already damaged by

disease at the start of treatment, and that makes it

difficult to harvest healthy stem cells. That is not a

problem here: the doctor can determine whether the

patient is at risk of developing dry mouth syndrome

before the radiation therapy actually starts. With the

help of a CT scan, the radiotherapist can predict

exactly which areas will receive how much radia-

tion. If it looks like there is a chance the salivary

glands will be damaged, the doctor can decide to

perform a biopsy on the salivary gland to extract

stem cells. These can then be cultivated while the

patient is receiving radiotherapy, so that they can be

placed back and do their work once the therapy has

finished.

“ It works in principle, but there are still some catches.”

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Microscope image of a salivary gland organoid from Rob Coppes’ lab (UMCG). The colours represent different types of cells, with the nuclei shown in blue.

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“We have already booked successes in mice”, says

Coppes. Mice whose salivary glands were damaged

by radiation had cultivated cells injected into their

salivary glands. Around one month later, the cells

had distributed themselves over the entire gland.

“You do not even need a microscope to see whether

the experiment succeeded. Mice clean themselves

with saliva, so a shiny fur clearly shows whether or

not their salivary glands are working properly.”

The cells do have to be reinjected in exactly the right

place. Salivary glands are shaped like a bunch of

grapes, Coppes explains: the stem cells are in the

‘twigs’, and the ‘grapes’ contain the active cells that

produce saliva. So the stem cells must get into the

twigs and spread from there, specialising into sali-

va-producing cells. In mice, it takes quite some

fiddling, but in humans you could easily check

this with ultrasound equipment.

There is still a long way to go. The fact that the prin-

ciple works in mice is a major but first step. Human

salivary gland stem cells that were transplanted into

mice with a suppressed immune system also did

their work. However, cell therapy is not freely

allowed, and the rules are strict. Coppes expects it

to take at least another eighteen months before the

first patient can be treated with this method. “The

growth medium has to be approved first, and you

have to be absolutely certain that you do not inad-

vertently inject tumour cells into the patient. It

works in principle, but there are still some catches.”

One bottleneck, for instance, is the medium used

for the cultivation of the cells. The current medium

is appropriate for animal cells, but has not been

approved for human cells yet; finding an approved

replacement has turned out to be difficult.

Apart from cultivating the stem cells, Coppes also

hopes to find a way to grow the entire gland. He is

looking for the correct growth factors and environ-

ment which allows salivary gland cells to grow into

an organ. He has succeeded in cultivating small,

two-millimetre salivary glands; in the future, he

would like to try to grow larger organs using each

individual patient’s own cells, which could then

be placed back into their body in their entirety and

might even be able to function straight away.

“ Whenever I eat a sandwich, I have to take a sip with each bite.”

Never eating toast again NIRM Case study

Some diseases are difficult to investigate simply because there is no research material. To solve this problem, Joost Gribnau, develop-mental biologist at the Erasmus University Medical Center, uses a clever trick. He creates stem cells (IPS cells) from patients’ skin cells and lets them mature into adult cells to make his own research material.

Gribnau conducted research into disorders where

X-chromosome inactivation plays an important role,

such as Rett syndrome. In a female foetus, one ran-

dom X-chromosome out of the two in each cell is

switched off during the embryonic phase. Therefore,

if there is a mutation in X, the foetus will have both

sick and healthy cells. It is the ratio between the

numbers of sick and healthy cells that determines

the extent to which the disorder becomes manifest.

Without growing his own research material, Gribnau

would not have been able to do his research. “We

wish to better understand and predict the selection

process in X-chromosome inactivation”, Gribnau

explains. Matured cells of patients were of not much

use to them. “The process of inactivation has already

come to an end in matured cells. So to go back to

the beginning of life we decided to make new cells

ourselves.”

A special facility was set up for the purpose of making

IPS cells from skin cells. The researchers can then

do all sorts of things with the stem cells they have

created. They can change them into brain cells, for

instance. This means that it should eventually be

possible to study and explain the behaviour of the

sick cells of each and every patient.

Rare“Unfortunately, the stem cells did not behave as

expected, so recently we have mainly focused on

pushing both X-chromosomes into action”, Gribnau

says. They have succeeded, so they can now finally

use these cells to research Rett syndrome and other

X-chromosome related disorders.

Gribnau's fundamental research is of great impor-

tance to patients. “We can now study disorders for

which there used to be no research material available,

such as brain diseases or very rare disorders. By

cultivating patients’ sick cells we gain not only a

better understanding of the process behind a disor-

der, but also an opportunity to test new medicines

or treatments.”

Make your own research material

The coat of a calico is a fine example of X-chromosome inactivation. The genes for coat color, red or black, in cats are coupled to the X-chromosome. In female cat embryos one of the two X-chromosomes is switched off. Sometimes the color is black switched off and sometimes red. This results in the familiar patch pattern.

story: Elles Lalieu image: Riosafari

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story: Elles Lalieu

Process report

2003

Th e project started with a simple

idea: take stem cells, turn them

into healthy heart cells, and inject

these directly into the heart mus-

cle. Th e objective: to prevent

heart failure in patients who just

had a heart attack, for instance,

and whose heart muscle is par-

tially defective.

2004

Our fi rst idea proved to be diffi -

cult to put into practice. Th ere

was no suitable lab animal at

hand. Th e mouse is not a good

model for cardiac disorders.

Th e heart of a mouse beats over

fi ve hundred times per minute,

a human heart only sixty times.

Moreover, the heart rate of a

mouse does not rise in stressful

situations, but a human’s does.

Th e heart of a pig would have

been a better model, but it is

rather diffi cult to suppress their

immune system; the stem cells

would quickly be rejected.

2007 Apart from the problem with the

lab animal, we now also had a

practical problem. Th e heart is

one great lump of muscle – how

do we get healthy heart cells into

the right places? Stem cells do

not travel and then settle exactly

where you want them to. I always

compare it to a piece of chewing

gum. If you inject red dye into it,

only a small part turns red, not

the entire piece. It works similarly

in hearts. Injecting separate cells

into the heart muscle was not a

success. A setback, but that is also

part of doing research. In any

case, the experiments taught us

quite a bit about the human heart.

To repair damaged organs: that is the great challenge in regenerative medicine. It was also the challenge that Professor Christine Mummery faced when she started to conduct research on the heart. Repairing the heart using stem cells proved more diffi cult than expected – so Mummery took a different tack.

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2010

To create a synthetic heart, we

fi rst had to be able to produce all

the cell types of the heart in the

lab. Th is sounds easy, but it was

an enormous challenge. Sixty per

cent of a foetal heart is made up of

beating heart cells, but in adults

this is only thirty per cent. More-

over, beating heart cells come in

diff erent types. Th e cells of the

atriums, for instance, look slightly

diff erent from the cells of the

ventricles, not to mention the

blood vessel cells that supply the

heart with oxygen, connective

tissue cells that ensure its solidity

and pacemaker cells that regulate

its rhythm.

2009

We started afresh with a new

goal: to make a synthetic piece

of heart. Of course, we did not

do this just for the sake of it: the

synthetic heart could be used as

a test model for the development

of drugs. We are not only able to

make heart cells from healthy

people, but also from patients

with heart disorders. We simply

isolate kidney cells from their

urine and treat them in the lab to

make them turn into stem cells.

And who knows, we might be

able to use these synthetic heart

pieces in the future to repair

damaged hearts.

2011

Together with engineers, we went

looking for the perfect matrix for

our stem cells. In the body, heart

cells grow on a soft substrate. Th is

is a good environment for the

cells, so we needed to reproduce

it in the lab. We let the cells grow

on synthetic polymer, which is

somewhat similar to silicon. Th is

polymer is placed on a chip with

spiral-shaped electrodes inside

and a vacuum underneath. Th e

spiral-shaped electrodes form a

kind of stretch system that makes

the cells come into action. Th e

heart rate can be adapted by

changing the vacuum. In this way

we can test the diseased heart

cells at rest, but also look at what

happens if a patient does sports,

for instance.

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2009

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Mummery’s approach is successful as a test model, but it cannot be directly applied to patients yet. Cell biologist Marie José Goumans adopted another tactic: she isolates stem cells from the heart itself and subse-quently tries to shape them into functional heart cells. Since the cells originate from the organ to which they will eventually be returned, this method is closer to patients.

“We retrieve the cells from the right atrium of the heart”, Goumans explains. “There is an area there that we refer to as the ‘heart’s appendix’. We do not really know what its function is, but it is always removed in open heart surgery. Instead of discarding this bit of ‘waste’, the surgeons now bring it to our lab.”

The removed piece of cardiac tissue contains stem cells, albeit only a few. “We isolate the existing stem cells, cultivate them until we have many more, and then use growth factors to try and grow them into heart muscle or blood vessel cells”, Goumans explains. “Together with Carlijn Bouten’s research group (see also Design your own heart valve, ed.) we have also looked into their willingness to differentiate if we stretch them a little, like they do inside a beating heart.” However, stretching alone has proved not to be enough for stem cells to develop into heart cells.

The cultivated heart cells were implanted in mice that recently had a heart attack, but this brought another problem. Human heart cells do not like to attach to a mouse heart, as this forces them to beat over five hundred times per minute, causing them to blow themselves up. Without good lab animal results, how-ever, it is not possible to test the cells in patients.

According to Goumans, the solution lies in combining her cells and Mummery’s test system. “We could make a synthetic piece of heart and use this to grow the cultivated stem cells.”

X-ray of an appendix (the worm-shaped pouch below the villi). There is a similar piece of tissue

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Cultivating cells from the heart’s “appendix”

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At the moment we only have a

test model, but I do think there

will be applications for patients in

the future. We could use a piece of

synthetic heart as a patch or plas-

ter on a damaged heart. Of course,

this will bring its own set of prob-

lems. A heart attack often leaves

damage throughout the heart,

whereas a patch would only be

placed on the outside. Would that

work, or should we attach the

patch somewhere else? What

about attaching it just below the

outer layer of the heart? Th at

would be comparable to putting

something just underneath

the peel of an orange, without

peeling the rest of the orange.

Repairing a heart is not easy,

but that does not mean that you

should not try.

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Our model was successful. We

also received a number of inter-

esting follow-up assignments.

Even the pharmaceutics devel-

oper GSK showed an interest.

However, the system is still not

good enough. In the development

of drugs, it is important to be able

to examine a great many condi-

tions in one comprehensive test.

Th is is done using plates with a lot

of compartments (wells). As it is,

the chip does not fi t into these

wells. Th is means that we are only

able to examine one condition at a

time. Downsizing the chips so

that they fi t is a matter of engi-

neering. To get there, we are now

collaborating with Delft Univer-

sity of Technology.

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Summer 2015

Time for a celebration: after more

than fi ve years, we managed to

make eight diff erent types of

heart cells and cultivate them in

the lab. We knew how heart cells

develop in an embryo, but fi gur-

ing out which signals are involved

in this process was quite a job.

Zebra fi sh and mouse embryos

came in useful here: in these ani-

mals you can switch off one “sig-

nal” at a time and see the eff ects

on the development of the heart.

If you switch one off and see that

pacemaker cells are no longer

made, for instance, you can add

this switched-off signal to the

stem cells in the hope that this

will stimulate them to actually

make pacemaker cells.

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The heart still has some secrets to reveal

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Repairing liver damage with the patient’s own cells

Using stem cells to repair body parts: the idea is

pretty obvious, since stem cells are also responsible

for small repairs in organs in healthy people. In fact,

it is the damage that triggers stem cells into action.

Stem cell research has been going on for several

decades, Clevers explains. “The most important pro-

teins that play a role in embryonic development – the

WNT proteins that pass on signals between cells –

were discovered in the 1980s. We found out that they

are crucial in the continued activity of mature stem

cells. If they are overactive, this will lead to cancer.”

Clevers started his stem cell research in the bowel,

which has the most active stem cells of all organs,

renewing its cells every four days. In 2009, Clevers’

group succeeded in cultivating stem cells that had

only been discovered two years before. Last year, the

researchers at Clevers’ lab and their Japanese col-

leagues proved that it is possible to reorganise these

cells into mini bowels and return those so-called

organoids into the damaged bowels of mice –

thereby taking another few steps towards the ulti-

mate goal of stem cell therapy. Clevers: “If you can

use stem cells to repair a sick or damaged organ, the

shortage of donors would be far less of a problem.

The patient’s own body would serve as a ‘donor’ to

help repair its own tissue.”

The treatment method is not exclusively for the

bowel, although its great regenerative capability

makes it ideal. Clevers and his colleagues are also

working on the use of the same method for the liver.

Millions of people worldwide suffer from chronic

liver failure, caused by a genetic defect or by viruses,

alcohol or other toxic substances. A liver transplant

can sometimes be helpful, but there are far too few

donor organs available. With the method that Clev-

ers’ group has developed within the NIRM consor-

tium, a simple injection would suffice to repair the

entire liver.

The liver is special, as it is usually able to repair itself

quite effectively. If you take away part of the liver, it

When a damaged liver is no longer able to repair itself, a transplant is the only option.

However, to the tens of millions of people with liver damage in this world, there are far

too few donors available. Hans Clevers, professor of Molecular Genetics at the Hubrecht

Institute of the KNAW Royal Netherlands Academy of Arts and Sciences, is developing a

technique to grow mini livers from stem cells that can make the organ as good as new.

“ It would be ideal if we could already start treating patients while they’re on the waiting list.”

story: Rineke Voogt

NIRM ?

may even grow back again. A sick liver has lost this

ability but would, in theory, only need a few trans-

planted healthy stem cells to regain its function.

Th is makes the liver – unlike, for instance, the heart

– very suitable for this type of research.

In January 2015, Clevers published a so-called proof-

of-concept study, based on precisely this idea, in the

scientifi c journal Cell. Th e researchers extracted

stem cells from the hepatic ducts of a donor liver,

and started cultivating them. “You will fi rst have to

make billions of cells with the help of the correct

growth factors”, Clevers explains. “If you have many

stem cells and precursors, they can specialise into

the two most important parts of the liver: hepato-

cytes (liver cells) and hepatic duct cells.” Th e liver

organoids made from those cells consist of tens of

thousands of cells and are only two to three millime-

tres in size, but they do have a real 3D structure.

Th e researchers placed these mini livers back into

mice with liver damage and a suppressed immune

system. Th e mice clearly recuperated: the healthy

cells replaced parts of their damaged liver.

“Although the liver damage in mice would have

been repaired in the long run anyway, we did see

that the mice treated this way recuperated more

quickly”, Clevers says.

In this stem cell therapy, the sick liver acts like a

scaff old, as regenerative specialists would call it. Th e

organoids need the infrastructure of the damaged

liver to attach themselves. Th is is relatively simple

in livers: “You can inject the organoids in the portal

vein, which transports blood directly into the liver.

Th e portal vein branches off into very tiny capillar-

ies, where the organoids simply get stuck and easily

colonise the liver.” Th e portal vein in mice proved to

be too small, so they received the mini livers in their

spleen. Even from there, the organoids could fi nd

their way into the liver and nest there.

It is a big step from lab animal research to humans in

this type of research. However, clinical experiments

with mature liver cells are already taking place, in

which billions of separate cells are administered

through the portal vein. Th is would be far more

eff ective with stem cells. But there are some catches.

Repairing liver damage with the patient’sown cells

Hans Clevers: “It would be ideal if we could already start treating patients with organoids while they’re on the waiting list for a transplant.”

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Technically, according to Clevers, researchers are

making good progress. They have managed to

upscale the cell cultivation in order to grow billions

of cells from just a few stem cells. Legislation is the

main problem. The biggest concern is the risk of

cancer in the tissue due to mutations in the organ-

oids. This risk seems to be nil, says Clevers: “We

don’t see that happening in cell cultivation. If you

maintain the optimal environment for the cells – add

the correct substances and ensure that the cells can

grow properly – you will avoid cancer. The cells are

genetically very stable.”

“It would be ideal if we could already start treating

patients with liver organoids while they’re on the

waiting list for a transplant”, Clever believes.

“But in order to do so, we still have to clear some

hurdles.” The researchers first want to know for

certain, for instance, how many organoids they

will have to administer for an optimal effect and

whether these should be stem cells or mature cells.

Clevers is confident that the principle also works in

humans. “There have been patients with a liver dis-

ease whose genetic defect was corrected in a single

cell, purely by chance. Little by little, the liver

repaired itself from that one cell, and the patients

regained their health. If a single healthy cell can

be enough to heal someone, treatment with mini

livers opens up a new perspective for millions of

patients.”

A microscope image of a liver organoid.

“ It would be like using the patient’s body as a donor.”

Repairing liver damage with the patient ’s own cells Case study NIRM Case study

Cell biologist Gerald de Haan at the University Medi-

cal Center Groningen found a solution. He equipped

cells with a “barcode” so that they can always be

identified. “We take stem cells from the bone marrow

of mice, to which we introduce a small piece of new

DNA”, he explains. “We then transplant the stem cells

back into the bone marrow. If one of these stem cells

starts to divide, all of its offspring share the same code

in their DNA. After transplantation we extract some

blood, isolate the DNA and then determine how many

times the barcode appears.”

The first results of barcoding, as the researchers call it,

are promising. In mice (which live to an average age

of eighteen months to two years), one or two stem

cells are responsible for 95 per cent of all the blood

cells formed during their lives. The same experiments

are now being repeated with human stem cells from

the umbilical cord.

Fewer lab animalsBarcoding not only provides insight into the forma-

tion of blood cells, but it also teaches us more about

blood cancer – leukaemia. “Not all leukaemia cells

are identical”, De Haan explains, “which means

that there is not just one single type of cancer in the

body, but a number of subtypes. Because we are now

able to recognise the stem cells, we can count how

many different stem cells are involved in a disease

like leukaemia and whether they respond differently

to treatment.”

An added advantage of barcoding is that research

now requires far fewer lab animals. Say, you have

one hundred stem cells and you want to know what

each of them does. De Haan: “Normally you would

transplant those one hundred stem cells into one

hundred different mice. Now you can equip the

stem cells with a barcode and transplant them into

a single mouse. This makes the study both cheaper

and more efficient.”

A piece of DNA (green) is placed in a viral vector – a kind of transport vehicle that can deliver DNA into the cells. Once it is inside the cells, the newly added DNA integrates into the existing DNA, which leaves all the cells with their own unique barcode (line patterns on the right).

Bone marrow contains stem cells that can make new blood cells. Once these new blood cells have entered the bloodstream, they are all identical and you can no longer recognise which blood cell originated from which stem cell. That is why we do not exactly know how many stem cells are involved in the formation of blood cells, nor if their number changes when we age.

Barcoded blood cells

story: Elles Lalieu image: Evgenia Verovskaya

RetroviralVector

34 35

NIRM Discussion

Are the regulations ready for regenerative medicine?

Many of the projects within NIRM were “proof-of-concept” studies:

studies to fi nd out if a proposed concept actually works. Technical

glitches have mostly been overcome, but the application in clinical

practice is a great step further. Are the regulations ready for it?

What must be done in order to make regenerative medicine available

to patients? Hans Clevers (Hubrecht Institute) and Carlijn Bouten

(Eindhoven University of Technology) explain.

NIRM

story: Rineke Voogt image: René den Engelsman

Regenerative medicine is not being used in practice to its full potential. Why is that?Clevers: “It is diffi cult to get approval for treatments

like stem cell therapy to repair a defect in the body.

If we succeed in repairing the liver of a mouse with

a mini liver, this is not a carte blanche to try out the

same approach in humans in a clinical experiment.

In a way this is understandable. After all, we are

working with living cells which, potentially, may

behave badly – you have to make certain that the

cell therapy that you want to use does not cause

cancer in the patient.”

So you have to provide evidence that the treatment is safe. How do you do that for a new therapy?Clevers: “Th is is precisely the problem. When is it

safe to apply a new technique? Th e cells that we

grow, to make liver organoids for example, are very

stable and do not mutate. Cancer emerges from

mutations. Th is is why the cultivated cells are, in

all probability, quite safe. However, even with the

large numbers of patients with a defective liver on

the waiting list, a transplant with liver organoids

cannot take place as yet. You will fi rst have to pro-

vide numerous examples to show that it is safe, but

it is in fact a catch-22: you will need clinical trials

to provide such examples.”

Why is it so diffi cult to test a treatment in a clinical trial?Clevers: “Th ere is great fear of repeating the thalido-

mide scandal (when a great many pregnant women

were administered this drug against morning sick-

ness and it was found to cause serious deformities in

their foetuses, ed.). Moreover, the pharmaceutical

industry is not very keen on cell therapy. It is easier

to produce and sell a treatment wrapped up in a pill;

living material is logistically complicated. Th is resis-

tance is an additional challenge in our work.”

Bouten: “As regenerative medicine is still a rather

new fi eld, many rules have not been fi xed as yet.

For example, for how many lab animals should you

provide evidence that your treatment works before

you are allowed to start a clinical trial? Th e therapy

is new, so there are no clear guidelines for each case.

As a researcher, therefore, you partly make your own

rules. For instance, in collaboration with doctors we

have decided that the heart valve for our study (see

“Design your own heart valve”, ed.) should work in

a lab animal for at least two years before we can take

the next step to humans. It is possible to accelerate

the same processes in the lab, but then the question

is whether all the safety aspects are covered and

whether the regulators would accept this.”

How do we get regenerative medicine from the lab into patients?Bouten: “Th e process of successfully developing a

new treatment from idea to patient usually takes

approximately fi fteen years. Th is might be even

longer for regenerative medicine, which is quite

diffi cult to fi t in with the daily clinical practice. It

is a great step from the lab to the market, as many

treatments are simply quite expensive. And once a

new method has fi nally reached the clinical phase,

the question is which patient group should be tack-

led fi rst. Doctors tend to prefer the group that runs

the least risk, but we have noticed that patients take

a completely diff erent view. Th at is an ethical issue

that needs further debate.”

What should change in order to make regenerative medicine more common?Bouten: “To fi nd a solution, we should look to the

new generation. Not everyone wants to go along

with a new treatment, even if it is already techni-

cally possible. We should introduce young doctors

to this new type of medicine and technological

progress at a very early stage of their training. Th is

will encourage them to start exploring the options

and adopt a more informed position. If you want to

apply regenerative medicine, you will need the sup-

port of the practitioner.”

Clevers: “Th e main challenge is to take away the fear

of a new treatment being administered without hav-

ing been properly thought out and researched – the

idea that it is bound to go wrong. Th is is precisely the

reason why we should also look beyond the medical

world: we need to keep society well-informed and

enter into public debate about the issue.”

3736

When something breaks down, you can try to repair or replace it – likewise in our body. But how do you actually repair a defective heart valve, an injured knee or severely burned skin? And, even more importantly, how do you repair it for the long term and without any side effects? These challenging questions are the focus of the Netherlands Institute of Regenerative Medicine (NIRM). Its research is divided into fi ve clusters: heart and blood vessels, muscles and bones, blood, nerve system and internal medicine.

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3938

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Contact: Ruud Bank, r.a.bank@umcg.nl, +31 50-3618043 Rini de Crom, nirm@erasmusmc.nl, +31 10-7043063

ColophonEditors

Joost van der Gevel, Elles Lalieu, Rineke Voogt

Chief editor

Sciencestories.nl, René Rector

Graphic design

Parkers, Rick Verhoog and Sara Kolster

Infographics

Parkers, Marjolein Fennis and Sara Kolster

Project leader

Giovanni Stijnen, NEMO Science Museum

Coordination

Giovanni Stijnen and Sanne Deurloo, NEMO Kennislink

This publication was realised with the financial support of the LSH-FES subsidy programme and in collaboration with NEMO Science Museum (publishers of NEMO Kennislink) and Marja Miedema and Miriam Boersema (NIRM).

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