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Stem cell technology

Paulo A Fontes, Angus W Thomson


Thomas E Starzl Transplantation Institute, Department of Surgery, University of Pittsburgh Medical Center,
4C Falk Clinic, Pittsburgh, PA 15213, USA
Paulo A Fontes
assistant professor of surgery. Angus W Thomson
professor of surgery and molecular genetics and biochemistry
Correspondence to: P Fontes fontesp@msx.upmc.edu
BMJ 1999;319:1308

Stem cell technology is a rapidly developing field that combines the efforts of cell biologists, geneticists, and clinicians and
offers hope of effective treatment for a variety of malignant and non-malignant diseases. Stem cells are defined as
totipotent progenitor cells capable of self renewal and multilineage differentiation.1 Stem cells survive well and show stable
division in culture, making them ideal targets for in vitro manipulation. Although early research has focused on
haematopoietic stem cells, stem cells have also been recognised in other sites. Research into solid tissue stem cells has not
made the same progress as that on haematopoietic stem cells. This is due to the difficulty of reproducing the necessary and
precise three dimensional arrangements and tight cell-cell and cell-extracellular matrix interactions that exist in solid
organs. However, the ability of tissue stem cells to integrate into the tissue cytoarchitecture under the control of the host
microenvironment and developmental cues, makes them ideal for cell replacement therapy. In this overview, we briefly
discuss the current research and the clinical status of treatments based on haematopoietic and tissue stem cells.

Summary points
Stem cells are progenitor cells that are capable of self
renewal and differentiation into many different cell lineages
Stem cells have potential for treatment of many malignant
and non-malignant diseases
Peripheral blood stem cells are used routinely in
autologous and allogeneic bone marrow transplantation
Gene transfer into haematopoetic stem cells may allow
treatment of genetic or acquired diseases
Embryonic stem cells may eventually be grown in vitro to
produce complex organs
Neuronal stem cells are being used for neurone
replacement in neurovegetative disorders such as Parkinsons
and Huntingdons diseases
Haematopoietic stem cells
Applications of cultured haematopoietic stem cells
Haematopoietic stem cells are a somatic cell population
with highly specific homing properties and are capable of self
renewal and differentiation into multiple cell lineages.2 Human
haematopoietic progenitor cells, like stromal cell precursors in
bone marrow, express the CD34 antigen, a transmembrane cell
surface glycoprotein identified by the My10 monoclonal
antibody.3 However, pluripotent stem cells constitute only a small
fraction of the whole CD34+ population, which is by itself rather
heterogeneous regarding phenotype and function. The best way
to define haematopoietic stem cells is from their functional biology. They are known to restore multilineage, long term haematopoietic cell differentiation, and maturation in lethally
cytoablated hosts.4 Haematopoietic stem cells can be obtained
from bone marrow, peripheral blood,5 umbilical cord blood,6 and
fetal liver.7
The use of peripheral blood stem cells in both autologous
and allogeneic transplantation has become routine as they can
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be collected on an outpatient basis and also promote a consistent acceleration in haematopoietic reconstitution after
engraftment.8 Umbilical cord blood stem cells have been used
progressively in paediatric patients, from both related and unrelated HLA-matched donors. In recipients with severe T cell
immunodeficiency disorders, fast engraftment is required
together with a low risk of graft versus host disease and a low
viral transmission rate.9 Since umbilical cord blood stem cells
can be expanded in vitro or frozen for storage in cell banks10 they
have been used in clinical trials for both autologous and allogeneic haematopoietic stem cell transplantation.11
The bone marrow is a mesenchyme derived tissue consisting of a complex haematopoietic cellular component supported
by a microenvironment composed of stromal cells embedded in
a complex extracellular matrix.12 This extracellular matrix has an
important role in the facilitation of cell-to-cell interaction, in
addition to a more complex role in the binding and presentation
of cytokines to the haematopoietic progenitor cells.13 The
cytokine milieu and extracellular matrix interaction provides the
road map for maturation and differentiation of stem cells,14
which should be instrumental for their in vitro manipulation
before therapeutic use. For example, haematopoietic stem cells
can be manipulated in vitro to generate dendritic cells, the most
potent antigen presenting cells.
Dendritic cells have a pivotal role in the elicitation and regulation of antigen specific, major histocompatibility complexrestricted T cell responses and are thought to be the only antigen
presenting cells able to prime naive T cells. Dendritic cells can be
derived from CD34+ precursors in response to granulocyte
macrophage colony stimulating factor and tumour necrosis factor and from monocytes cultured with granulocyte macrophage
colony stimulating factor and interleukin-4.15 In vitro generated
dendritic cells (fig) that have been transduced with genes coding
for tumour specific antigens or pulsed with tumour specific antigen or peptide could be useful for induction of cytotoxic T cell
responses.16 Dendritic cell tumour vaccines could be important
1

Characteristic dendritic cells derived from CD34+ haematopoietic stem


cells and propagated in granulocyte macrophage colony stimulating factor

future therapeutic tools; phase II clinical trials are under way and
show limited efficacy.17 On the other hand, the migration and
function of dendritic cells derived from liver in an allogeneic environment may be seminal in the development of donor specific
tolerance.1820 Genetic engineering of dendritic cells to express
immunosuppressive or immunoregulatory molecules may
provide a novel method to promote graft tolerance, reducing
dependence on systemic immunosuppression.21
Haematopoietic stem cells and gene therapy
Haematopoietic stem cells themselves are also a promising
target for gene therapy. Haematopoietic stem cells have been
made resistant to one or more cytotoxic drugs22 with retroviral
transfection of the multidrug resistance gene (MDR1). This
should help circumvent the myelosuppressive effects of standard
regimens of chemotherapy.23 Haematopoietic stem cells can also
be genetically marked to allow assessment of patterns of cell
survival, localisation, and function after bone marrow transplantation.24 This strategy has already been used with retroviral vectors.25 Double genetic marking is also being used to determine
the long term effects of different protocols of cytokines given to
promote bone marrow regeneration after cytoablative treatment.26 Gene transfer into haematopoietic stem cells represents
a novel approach for treating some genetic or acquired diseases.
So far, transduction of genes into humans using haematopoietic
stem cells has shown low efficiency, especially in the quiescent
stem cell population.27
Recent advances in reproductive biology and gene therapy
have used ex vivo transduced autologous umbilical cord blood
cells or direct targeting in utero as a potential means to correct
haematopoietic, immunological, and metabolic single gene disorders.28 This technique has the advantage of using normal haematological development, which induces the fetus to allow
space for a new cell population and promotes tolerance in the
developing immune system. Unfortunately, infection and graft
versus host disease are still potential risks for both the mother
and the fetus. However, advances in the understanding of dose
requirements and manipulation of peripheral blood sources to
enrich for stem cells may provide strategies to overcome these
problems.
Non-haematopoietic stem cells
The adult bone marrow also contains mesenchymal stem
cells29 which are involved in the regeneration of mesenchymal
tissues such as bone, cartilage, muscle, ligament, tendon, adipose tissue, and stroma. Although human mesenchymal stem
cells have been isolated, it remains unclear how basal nutrients,
cell density, spatial organisation, mechanical forces, growth factors, and cytokines control their differentiation.30 Isolated human
mesenchymal stem cells constitute a single, phenotypically distinct population and are uniformly positive for SH2, SH3, CD44,
CD71, CD90, CD106, CD120a, and CD124. They are also
2

negative for CD14 and CD34 and can be induced to differentiate


into adipocytes, chondrocytes, tenocytes, and osteocytes.
Transplantation of mesenchymal stem cells into tendon defects
in rabbits can significantly improve biomechanical properties of
the damaged area.31
Embryonic stem cells were first isolated in 1981 through
studies focusing primarily on murine blastocysts.32 Embryonic
stem cells are derived from totipotent cells of the early mammalian embryo and are capable of unlimited, undifferentiated proliferation in vitro.33 Human embryonic stem cells can express high
levels of telomerase activity, comparable with that expressed by
cells isolated from germ lines and embryonic tissues.34 They can
also form several cell types and simple tissue. Further
understanding of cell tissue interactions and their relation with
the extracellular matrix may eventually enable in vitro production
of complex organs.35 In vitro manipulation of embryonic stem
cells can be enhanced by nuclear transfer and cloning.36
Advances in stem cell technology have stimulated rapid
growth in the understanding of embryonic and postnatal neural
development. A population of neuronal stem cells capable of
extended self renewal in addition to subsequent differentiation
into both neurones and glia has already been identified.37 These
common neurohaematopoietic stem cells can be isolated from
the subventricular zone in the wall of the lateral ventricle of the
brain. They divide in response to epidermal growth factor and
fibroblast growth factor-2.38 Transplanted neuronal stem cells
can integrate into an intact brain and differentiate into neuronal
and glial cells. They may also function as haematopoietic stem
cells when infused into irradiated mice.39 This last finding is controversial and may represent an artefact of the experimental
technique, since only one contaminating haematopoietic stem
cell would be needed to repopulate a mouse haematopoietic
system. However, the reverse has also been shown, with stem
cells derived from bone marrow entering through the brain-blood
barrier, becoming fully differentiated, and displaying
macrophage-like function (microglia).40
Initial clinical trials have shown that neurone replacement
for neurovegetative diseases such as Parkinsons and
Huntingtons diseases is now feasible. Reports of transplantation
of fetal striatal tissue in patients with mild to severe Huntingtons
disease suggest that transplantation of neuronal stem cells may
improve some of the cognitive symptoms associated with the
condition, as well as potentially modifying its clinical course.41
Neuronal stem cells can also be manipulated before grafting;
they have been shown to respond to stimulation with fibroblast
growth factor-2, and immortalised neural stem-like cells infected
with viral vectors have been found to express factors that are
related to neural repair.42
The existence of prostate stem cells is still a matter of
debate.43 Nevertheless, the concept is interesting, mainly
because of the possible analogy between effects of androgens
on the prostate stem cells and that of cytokines on haematopoietic stem cells. Prostate stem cells could have important
implications in the development of prostatic carcinoma.44
Understanding of liver regeneration has improved greatly
since the initial description of oval cells as progeny of facultative
stem cells.45 Hepatic stem cells, which are found in the
interlobular bile ducts and possibly also in the canals of Hering,
could have a large impact on the pathophysiology of hepatocellular carcinomas and cholangiocarcinomas.46 47
Limbal stem cells have been described at the junction
between the cornea and sclerae.48 These cells are known to be
progenitors of corneal epithelium. Keratolimbal allografts are a
promising treatment for bilateral blindness,49 and limbal stem
cells can now be safely obtained from living related donors.50
Conclusions
Stem cell technology is progressing as the result of
multidisciplinary effort, with clinical applications of manipulated
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stem cells combining developments in transplantation and gene


therapy. There are rather complex ethical issues related to the
applications of cloning and nuclear transfer in human stem cells.
Successful ex vivo manipulation of stem cells will depend on
improved understanding of the interactions between cytokines
and the extracellular matrix. Cytokines may decrease binding
forces between stem cells and components of the stromal
microenvironment, thus facilitating the migration of stem cells
into the peripheral blood. Improvements in mobilisation
schedules using growth factors, stem cell isolation, and purification procedures and techniques for both positive and negative
purging (to reduce tumour cell contamination or to deplete T
lymphocytes) are emerging. The possibility of ex vivo multiplication of stem cells to accelerate haematopoietic recovery or to
provide sufficient stem cells from one extraction to support
several cycles of high dose chemotherapy is under investigation.
The applications for autologous stem cell transplantation should
increase as it avoids the use of non-specific immunosuppressive
therapy. Peripheral blood stem cells have advantages over bone
marrow cells for autologous transplantation in that they show
consistently faster haematopoietic reconstitution and can be
collected on an outpatient basis.
Stem cells originating from solid tissue can potentially be
applied in tissue repair. Techniques by which new genetic material is introduced into stem cells are being developed, and may
lead to the cure of various inherited diseases by somatic gene
therapy.
We thank David Kramer, Dana Faratian, and Dona Kocan for
scentific help with preparing the manuscript.
Funding: NIH grants DK49745 and AI41011.
Competing interests: None declared.
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