J R Soc Med 2005;98:346-350
doi:10.1258/jrsm.98.8.346
© 2005 Royal Society of Medicine
Embryonic stem cells and tissue engineering: delivering stem cells to the clinic
A Vats FRCS 1,2
N S Tolley FRCS 2
A E Bishop PhD 1
J M Polak DBE FRCPath 1
1 Tissue Engineering and Regenerative Medicine Centre, Faculty of Medicine,
Imperial College London, Chelsea & Westminster Hospital, 369 Fulham Road,
London SW10 9NH, UK
2 St Mary's Hospital, Praed Street, London W2 1NY, UK
Correspondence to: A Vats E-mail:
a.vats{at}imperial.ac.uk
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INTRODUCTION
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One of the challenges facing biomedical science is of its own
making—namely,
the progressive increase in lifespans. An increasing
number
of individuals require treatment and the treatments themselves
have to
work for longer. A shift in emphasis is therefore required
towards biological
approaches including the regeneration of
tissues. Tissue engineering can be
best defined by its goal—the
design and construction in the laboratory
of living components
that can be used for the maintenance, repair or
replacement
of malfunctioning tissues. In this discipline, born in 1933
when
tumour cells were wrapped in a polymer membrane and implanted
into a
pig,
1 the life
sciences and medicine come together with
engineering in activities centred on
three basic components—cells,
scaffolds and signals. The development of
tissue engineering
has lately been spurred by the increased availability of
cell
sources, proteomics, the advent of new biomaterials, improvements
in
bioreactor design and increased understanding of healing.
However, neither
commercial development nor clinical application
of tissue engineered products
has kept pace with this rapidly
evolving research. Industrial development has
been hindered
by difficulties in devising cost-efficient processes,
guaranteeing
product viability and satisfying the regulators. Nevertheless,
the
coming years will see a large increase in the number of patients
benefiting
from tissue engineering. For the cell biology component of tissue
engineering,
the greatest challenge is to optimize the isolation,
proliferation
and differentiation of cells and to design scaffolds or delivery
systems
that yield tissue growth in three dimensions. Ideally, we would
harvest
stem cells from a patient, expand them in cell culture, seed
them on a
scaffold and then implant the resultant tissue. Stem
cells, when given the
specific biological stimuli, can differentiate
to become many types of
specific mature cells, and use of these
cells avoids the immunorejection that
can occur with donor transplants.
In addition, the technique of somatic
nuclear transfer allows
the creation of autologous cells and tissues from
allogeneic
embryonic stem cells. It is then important for the scaffold
to act
as a template and stimulus for proliferation and differentiation
of the stem
cells into the mature cells that will generate specific
new tissue. The tissue
can be grown on a scaffold that will
resorb, so that only the new tissue will
be implanted, or a
'biocomposite' of the scaffold and new tissue can
be implanted.
After implantation, the tissue-engineered construct must be
able
to survive, restore normal function and integrate with
the surrounding
tissues.
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CELL SOURCES
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The success of tissue engineering depends on the generation
of appropriate
cells and the ability of those cells to perform
specific biological functions.
For example, cells must produce
extracellular matrix in the correct
organization, secrete cytokines
and other signalling molecules, and interact
with neighbouring
cells/tissues. Tissue engineers have, over the years, looked
at
virtually all tissues in the body. In some cases, it has been
possible to
repair/replace tissue using, as a starting material,
the relevant cells from
the same patient—such as knee
repair with autologous
chondrocytes.
2
Non-specific cell types
have also been used, including dermal fibroblasts for
heart
valve
engineering.
3 These
early cell sources had severe limitations,
including low yield and the
possibility of gene alterations
related to age. Although primary (especially
autologous) cells
are still used in tissue engineering, stem cells constitute
an
important new resource.
Stem cells are commonly defined as undifferentiated cells that have the
capacity both to self-renew and to differentiate into one or more types of
specialized cells; however, this definition has required reconsideration in
view of the observation of dedifferentiation and transdifferentiation of
certain mature
cells.4–6
Therefore, some workers now favour a broader definition, applicable to a
biological function that can be induced in a range of cell types, including
differentiated cells, rather than a single
entity.7 Current
sources of stem cells for tissue engineering include embryos and adult donors.
The range of cell types to which they can differentiate varies, with embryonic
stem cells the most pluripotent. For tissue engineering, stem cells can
provide a virtually inexhaustible cell source. Current research is focused on
promoting stem cell differentiation to required lineages, purification of
consequent cells, confirmation that there is no residual carcinogenic
potential in the cell population and implantation in a form that will replace
or augment the function of diseased or injured
tissues.8,9
Both types of stem cell, embryonic and adult, have drawbacks. With embryonic
stem cells there are ethical considerations, together with the possibility of
tumorigenicity; also, not many cell lines are available. Adult stem cells are
more limited in potential and are often difficult to harvest in sufficient
numbers. Thus, the search continues for an ethically non-controversial, easily
accessible and abundant source of stem cells. The discussion here focuses on
embryonic stem cells.
Embryonic stem cells
Pluripotent stem cells can be divided into three types—embryonic germ
cells derived from primordial germ cells, embryonic stem cells and embryonic
carcinoma cells. The usual view is that these cells are able to differentiate
into all cells that arise from the three germ layers but not the
embryo; however, this dogma has been challenged by the discovery that
embryonic stem cells can differentiate to trophoblast cells in
vitro.10
The identification and isolation of embryonic stem (ES) cells from the
mouse, by Evans and Kaufman, was a major event in
biology.11,12
From the early 1950s, the work of Stevens and Pierce (reviewed by
Alexandre13) had
shown that teratocarcinomas contain cells with multilineage potential. The
isolation and culture of embryonal carcinoma (EC) cells gave developmental
biologists an in-vitro model with which to study the processes of
differentiation.14
The isolation of ES cells from first
primate15 and then
human blastocytes16
was the step that put these cells at the forefront of regenerative medicine
research. The huge promise of this approach is indicated by the spate of
papers that soon followed, recording differentiation of human ES cells to
neural cell types (neurons, oligodendrocytes and glia), cardiomyocytes, beta
cells, osteoblasts, hepatocytes and haematopoietic
progenitors.17–23
One intriguing possibility, the use of somatic nuclear transfer to create
autologous ES cells for therapy, has been widely discussed since the creation
of the first cloned
animal.24 Recently,
Hwang et al. have shown that this technique can be applied to human
oocytes, resulting in blastocyst formation and derivation of an ES cell
line.25 The
shortage of human ES lines currently available and restrictions in some
countries on human ES cell research prevent the whole scientific community
from participating in this field. The announcement that seventeen new human ES
cell lines will be made freely and widely
available26 is a
great fillip to researchers, though therapeutic use will have to await
development of clinical-grade cell lines and resolution of the ethical
debates.
Genetic and epigenetic changes can occur following multiple passages of ES
cell in culture.27
Self-renewal and also simultaneous suppression of the differentiation of ES
cells is ensured by a unique network of transcription factors including Nanog,
OCT4 and Wnt. Nanog was discovered by expression cloning analysis and named
after the mythological Celtic land of the ever young, Tir nan
Og.28 Oct4, or
octa-binding factor 
, is a member of the Pit-Oct-Unc family of
transcriptional regulators restricted to early
embryos.29 The Wnt
family consists of secreted and extracellular-matrix-associated glycoproteins
binding to frizzled seven-transmembrane span
receptors.30
Recently, DNA
microarray,31,32
SAGE33 and cDNA
library analysis34
has enabled the identification of global transcription profiles for human ES
cells. All demonstrated the existence of gene clusters that are expressed at
higher levels in human ES cells than in fully differentiated cells. Human ES
cells are characterized by their expression of SSEA3, SSEA4, TRA-1-60 and
TRA-1-81 antigens that are now known to be down-regulated during
differentiation while several other antigens are
induced.34
In order to accomplish the transition of human ES cells in the laboratory
to clinical application, efforts have been focused on defining the culture
conditions needed to derive specific cell phenotypes and their progenitors.
The aim is to obtain cells in numbers sufficient for implantation and in
conditions of 'good medical practice' (i.e. uncontaminated by
foreign cells). The potential hazard of tissue rejection also has to be
addressed. In the UK the Medical Research Council has already established a
stem cell bank that will provide cell lines to match patients'
requirements. Somatic nuclear transfer, or therapeutic cloning, has been used
to generate animals with a common genetic
composition.35 For
therapeutic purposes, a nucleus would be taken from one of the patient's
somatic cells and transferred into an enucleated donor oocyte, then ES cells
would be isolated from the inner cell mass of the cloned embryo; these cells,
when implanted, would not be
rejected.25
Society's current perception of the value and medical potential of ES
cells has been influenced by a mixture of media reportage, religion and
politics. In some countries, a bias towards research on adult rather than
embryonic stem cells arises from objections to the destruction of human
embryos necessary for derivation of ES; but in the UK the Human Embryology and
Fertilization Authority has authorized the use, and in some instances the
creation, of human embryos for therapeutic purposes. Research into adult and
embryonic stem cells is not sufficiently advanced for a definitive judgment on
whether one source is better than the other as a basis for developing a broad
range of stem cell therapies. Each is likely to have its own niche in therapy,
and for some conditions a combination of both may prove best.
Various protocols have proved effective for driving the differentiation of
ES cells to particular lineages, including the use of growth factor
supplementation of media and genetic modification. In our own centre a robust
system has been developed for
osteoblast36,37
and
pneumocyte38–40
differentiation from murine and human ES cells employing a defined culture
medium. Cell-type-restricted promoters driving expression of either antibiotic
resistance genes or
fluorophores41 make
it is possible to separate the desired cell types as the ES cells
differentiate. RNA interference to knock down gene expression in ES cells is
another advance that produces enriched populations as well as helping
elucidate gene function in early
development.42,43
The ability of cells to interact with stromal cells and repopulate the
corresponding niches—a property known as homing—is essential to
the success of stem cell transplantation. Signalling molecules such as
chemokines and growth factors are thought to be involved in the mechanisms
regulating homing and the expression of these increases following tissue
injury. Co-culture of embryonic and other stem/progenitor cells with mature
cells or tissues is being used increasingly to drive differentiation towards
required
lineages.44,45
Our recent work showed that co-culture with murine embryonic pulmonary
mesenchyme is an efficient means to upregulate the differentiation of ES cells
towards
pneumocytes.46
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THREE-DIMENSIONAL INTERACTIONS
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The normal function of most cells and tissues depends not only
on soluble
factors but also on spatial interaction with neighbouring
cells and with a
substratum or extracellular matrix (ECM). Cell-cell
and cell-ECM interactions
are coordinated by several families
of membrane-spanning proteins known as
adhesion molecules. These
are fundamental to cell adhesion, helping to define
3-dimensional
cellular organization and also directly participating in cell
signalling
and controlling cell recruitment, growth, differentiation, immune
recognition
and modulation of inflammation.
Tissue engineering scaffolds have a dual purpose—to direct
morphogenesis in vitro and to maintain the structure and function of
the construct as it is integrated with the host tissues after implantation.
Various natural and synthetic materials have been used to produce
3-dimensional scaffolds to function as an artificial ECM. Scaffolds for tissue
repair ideally should be non-toxic, act as templates for tissue growth in
three dimensions, have good biocompatibility, be biodegradable, be able to
influence the genes in stem cells to enhance differentiation and proliferation
of all the phenotypes required for tissue regeneration, and be capable of
interacting specifically with the cell type(s) of
interest.47 Work
with such materials has shown how scaffolds can also be made bioactive through
adsorption with biomolecules, enabling recruitment and adhesion of specific
cell
types.48–50
For routine therapeutic use they will need to conform to regulatory standards
and be producible at reasonable cost.
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STRATEGIES FOR CLINICAL APPLICATION
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Most clinical applications of tissue engineering must still
be regarded as
experimental,
51
although skin substitutes and
cartilage grafts have been used successfully for
several years.
At present, the only reliable cell source for these is
autologous
cells from the patient. This source has serious limitations
in
terms of numbers of cells and generation of a viable extracellular
matrix.
Cloned, immortal, cell lines are capable of proliferating
but usually lack the
differentiation needed for stable tissue
repair. All tissues and organs have a
complex interdependence
of cell types with an inter-connected 3-dimensional
architecture.
Most tissue-engineered constructs involve only one, or at most
two,
cell phenotypes grown primarily in a 2-dimensional configuration.
This
compromise in structure limits clinical viability. All
tissues/organs have an
interpenetrating network of blood vessels
to provide nutrition and eliminate
waste products. Tissue-engineered
constructs at present lack this vital
network when they are
transplanted. The host tissues must quickly infiltrate
the tissue-engineered
graft with a blood supply or the cells will die. A major
challenge
is to achieve angiogenesis rapidly after implantation and maintain
a
viable nutrient supply as the construct becomes integrated.
Other issues encountered include the maintenance of sterility of a
tissue-engineered construct. Most methods used for sterilization of non-living
implants and devices, such as gamma-irradiation or autoclaving, kill cells.
Sterility must be achieved continuously up to the moment implantation is
complete. All of the above factors add to the manufacturing costs and at
present limit many tissue engineering applications to exploratory cases.
Moreover, since the long-term survivability of tissue-engineering constructs
is uncertain, ethical and legal considerations often require restriction to
patients in whom no other procedure is available. Use in these
'last-ditch' circumstances makes the viability and success of the
new procedures hard to assess. Finally, tissue-engineered products are subject
to the same regulatory procedures as non-living biomaterials and devices. At
present, only a few products have met these regulatory requirements. Costs and
risk/benefit factors are often hard to predict because of the uncertainty of
regulatory approval. Further development of tissue engineering, from bench to
bedside will be crucial in meeting the healthcare needs of the coming
century.
Note This paper is based on the Ellison-Cliffe Lecture, given at
the RSM on 19 October 2004 by Dame Julia Polak.
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TOP
INTRODUCTION
CELL SOURCES
