Download Stem cells: you can`t tell a cell by its cover

Human Reproduction Update, Vol.9, No.1 pp. 25±33, 2003
DOI:10.1093/humupd/dmg001
Stem cells: you can't tell a cell by its cover
Ian Rogers and Robert F.Casper1
Division of Reproductive Sciences, Fran and Lawrence Bloomberg Department of Obstetrics and Gynecology, Samuel Lunenfeld
Research Institute, Mount Sinai Hospital and The University of Toronto, Toronto, Ontario, Canada
1
To whom correspondence should be addressed at: Division of Reproductive Sciences, Department of Obstetrics and Gynecology,
University of Toronto, and Samuel Lunenfeld Research Institute, Room 876, Mount Sinai Hospital, 600 University Avenue, Toronto,
Ontario. E-mail: [email protected]
Embryonic stem (ES) cells are capable of unlimited self-renewal and have the ability to give rise to all tissue types in the
body. The use of human ES cells for tissue and cell therapeutics has been suggested, but is limited by ethical concerns as
these cells are derived from the inner cell mass of human embryos. In addition, the need for HLA matching of ES cellderived tissues for allogeneic transplantation would require a bank of several thousand ES cell lines to make tissue
therapeutics practical. Recently, adult stem cellsÐof which those in bone marrow are the best studiedÐhave been shown to
be capable of multilineage differentiation into cells of various non-blood tissues. Umbilical cord blood (UCB)
haematopoietic stem cells have been shown to be equivalent to bone marrow stem cells for reconstitution of the
haematopoietic system. Preliminary studies have also demonstrated that UCB haematopoietic stem cells are multipotent and
capable of differentiating into non-blood cell types. This observation raises the exciting possibility of replacing human ES
cells for tissue and cell therapeutics with UCB blood haematopoietic stem cells that are normally discarded with the
placenta after delivery.
Key words: adult stem cells/bone marrow stem cells/therapeutic cloning/transdifferentiation/umbilical cord stem cells
Introduction
Stem cells are de®ned as cells capable of unlimited self-renewal
and with the ability to give rise to multiple tissue types (Thomson
et al., 1998). Both of these parameters are subject to wide
interpretation and depend to some degree on whether the stem cell
is present in situ (in its normal environment) or in an experimental
setting. For example, embryonic stem (ES) cells, derived from the
inner cell mass (ICM) of mammalian embryos, have unlimited
self-renewal properties and give rise to all embryonic tissue types
in vitro, although unlimited self-renewal is not a property of cells
of the ICM in situ, where they differentiate into various tissues of
the body and the ES cell phenotype is lost. On the other hand,
adult stem cells, of which haematopoietic stem cells (HSC) are
the best studied, give rise to a wide range of progenitor and
mature cells within the con®nes of the haematopoietic system,
and have self-renewal properties for the life of the organism
(Harder et al., 2002). It is believed therefore that HSC have a
capacity for self-renewal but a more limited differentiation range
than ICM-derived cells. However, this view is now changing, as
described in the present review.
Experimentally, ES cells can be considered a `blank slate'
capable of responding to external signals from surrounding cells
or from exogenously added growth factors. The self-renewal
ability of ES cells and their capacity to differentiate into a wide
Ó European Society of Human Reproduction and Embryology
range of cell types make them ideal candidates for tissue therapy
(Amit and Itskovitz-Eldor, 2002). This novel use of human ES
cells has, however, created a moral and ethical dilemma (Green,
2001). There is no doubt that human ES cells can eventually
realise their clinical potential, although it is believed that the need
to derive these cells from a human embryo will always limit their
widespread use in tissue and cell therapeutics. The numbers of
spare human embryos are limited, not only by their availability
for research but also by their recovery rate after freezing. A
secondary problem limiting the use of human ES cells is the
required HLA matching of the derived cells or tissues to the
recipient, requiring a bank of several thousand ES cell lines to
make tissue therapeutics or gene therapy practical (Trounson,
2001).
The exciting question at present is whether tissue-speci®c stem
cells (also called adult stem cells) also have a `blank-slate'
phenotype. The recent demonstrations of bone marrow cells
giving rise to other tissues such as muscle (Hakuno et al., 2002),
neural (Meletis and Frisen, 2001; Zhao et al., 2002) or multiple
cell types depending on their experimental environment, support
this hypothesis (Krause et al., 2001; Jiang et al., 2002). The
identi®cation of mesenchymal-like cells within umbilical cord
blood (UCB) (Erices et al., 2000) suggests the possibility of using
UCB stem cells in the same manner. UCB stem cells have the
expected, and already demonstrated, potential to replace bone
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I.Rogers and R.F.Casper
marrow for reconstitution of the haematopoietic system
(Gluckman et al., 2001), but may also have the exciting
possibility of replacing ES cells for tissue therapeutics and gene
transfer in the future. In this regard, UCB stem cells have an
advantage over other types of stem cells as they can be collected
non-invasively at the time of birth from normally discarded tissue
Their use circumvents the ethical problems associated with the
creation of multiple human ES cell lines. The present review will,
therefore, focus on the potential of adult-derived stem cells,
especially UCB haematopoietic stem cells, as an alternative to ES
cells.
Embryonic stem cells and primordial germ cells
ES cells were originally derived from the ICM of the murine
blastocyst (Evans and Kaufman, 1981; Martin, 1981; Bradley et
al., 1984). These cells represent the ultimate in stem cells because
of their abilities to be both self-renewing and multipotent.
Chimeric analysis using murine ES cells has demonstrated their
capacity to contribute to all tissues (Nagy et al., 1990). Murine ES
cells can be maintained in vitro in a multipotent state (Wiles and
Johansson, 1999) and induced to form embryoid bodies which can
be differentiated into multiple adult cell types. Murine ES cells
have been used as a tool for the study of embryo development,
cell±cell interactions, the unscrambling of biological pathways,
and to derive models of human disease (Roach et al., 1995).
Furthermore, ES cells in which gene alterations have been made
can be manipulated in vitro and provide a constant source of cells
for study.
Some cell types will form spontaneously (cardiac myocytes and
hepatocytes), but others require an induction step using growth
factors (Jones et al., 2002). For example, during embryoid body
growth, vascularization will occur spontaneously but the addition
of both ®broblast growth factor (FGF) and vascular endothelial
growth factor (VEGF) or endothelial growth supplement to the ES
cell cultures enhances the production of endothelial cells (Vittet et
al., 1996; Balconi et al., 2000). Flk1+ ES cells can be
differentiated into blood, endothelial and smooth muscle cells,
thus recapitulating the normal embryonic development of the
vascular system that occurs in the blood islands of the yolk sac
(Yamashita et al., 2000). ES cells grown in similar conditions and
used to generate osteoblast cells from bone marrow aspirates will
generate osteoblast cells that will form bone nodules containing
calcium deposits. Spontaneous nodules form from embryoid
bodies without bone-enhancing factors, but the frequency is
greatly enhanced with the addition of phosphate, dexamethasone
and ascorbic acid (Buttery et al., 2001).
Both murine and human pluripotent stem cells have been
derived from primordial germ cells (PGC). Similar to ES cells
derived from the ICM, PGC stem cells (EG cells) will form
embryoid bodies capable of expressing markers representative of
all three germ layers: endoderm, ectoderm and mesoderm
(Shamblott et al., 1998). Different EG cell lines have been
shown to form embryoid bodies at a low frequency (Shamblott et
al., 2001), and the cells derived from the embryoid bodies could
be induced to express markers representative of haematopoietic,
vascular endothelial, neural, endoderm and muscle cells.
Furthermore, low-passage cells were compared with high-passage
number cells for levels of gene expression with no apparent
26
differences. This suggests that these lines can be stably
maintained and have properties similar to stem cells derived
from the ICM of blastocysts.
Human ES (huES) cells have now been found to exhibit
properties similar to those of murine stem cells, but with some
minor differences. Both require leukaemia inhibitory factor (LIF)
and feeder layers in order to maintain an undifferentiated state,
but some huES cells require the addition of bFGF as LIF is not
suf®cient to maintain the cells in an undifferentiated state (Amit
et al., 2000; Odorico et al., 2001). HuES cells have been tested for
gene expression by PCR, and have been grown in speci®c cultures
in order to determine their ability and range of tissue formation.
Removal of LIF from huES cells causes them to form embryoid
bodies, which will cavitate to form a blastocyst-like mass and
acquire characteristics of the endoderm, mesoderm and ectoderm.
These huES cells can form beating myocytes, neurone-like cells
and haematopoietic cells (Itskovitz-Eldor et al., 2000; Schuldiner
et al., 2000; Kehat et al., 2001). Although the clinical therapeutic
properties of ES cells are recognized to be immense, their origin
leads to ethical controversy. In addition, the requirement for HLA
matching with a recipient will result in the necessity of ES cell
banks or therapeutic cloning.
Therapeutic cloning
Although cloning has been successful in numerous mammalian
species, ef®ciency is low and surviving offspring appear to have
both physical and biochemical problems. For correct development
to occur, the genome undergoes a cycle of stage-speci®c changes.
During normal gametogenesis there are established differences
between the maternal and paternal genome at the time of
fertilization. Spermatogenesis results in the remodelling of the
chromatin with protamines, resulting in a tight, compact,
transcriptionally silent nucleus, whereas the oocyte contains
histones and an oocyte-speci®c linker histone. The spermatozoon
DNA is more methylated than the oocyte but becomes rapidly
demethylated soon after fertilization. The oocyte is slowly
demethylated due to DNA replication during cell cleavage. At
the blastocyst stage, the embryonic genome is under-methylated
and at this point identical de-novo methylation occurs in all DNA.
Exceptions to this uniform methylation are the imprinted genes,
which are marked during gametogenesis (for a review, see
Rideout et al., 2001) and may or may not be appropriately
expressed in cloned embryos.
Interestingly, nuclear transfer experiments have illustrated that
only in rare cases is the recipient oocyte able to reprogramme the
genome, presumably by restoring pre-zygotic methylation and
chromatin pattern. Although the exact mechanism of reprogramming has not been elucidated, studies with Xenopus have
suggested that cytoplasmic proteins of the recipient oocytes play
a crucial role (De Robertis, 1979).
The failure of nuclear transfer (NT) embryos is most likely due
to the common inability to reprogramme the genome effectively
so that genes required for correct development are reactivated in
correct temporal and spatial order. This is dif®cult, and the
probability of faithful completion is low. The majority of NT
clones in mammalian species fail to develop to term, and of those
that make it to term many die shortly after birth or develop other
Stem cells: you can't tell a cell by its cover
health-related problems (McCreath et al., 2000; Tamashiro et al.,
2002; Wilmut, 2002).
There are marked differences in success rates of NT with
somatic cells versus ES cells. Survival to the blastocyst stage is
dependent on the donor nucleus being in G0 or G1 phase. Whereas
most ES cells are in S-phase and most somatic cells are in G0 or
G1 phase, there is a higher blastocyst rate with somatic cells, but
most of these embryos do not survive to term. In contrast, of the
ES-derived NT embryos that reach the blastocyst stage, a greater
percentage survives post term. This observation suggests that the
G0 or G1 ES cells which result in blastocysts are better suited for
complete nuclear reprogramming (Campbell et al., 1996;
Wakayama et al., 1998, 1999; Wakayama and Yanagimachi,
1999; Rideout et al., 2000).
Recently, the utility of therapeutic cloning combined with gene
therapy was demonstrated in the mouse. An ES cell line was
derived from Rag2±/± immunode®cient mice and the genetic
defect was corrected in the ES cells with a gene insertion. The
corrected ES cells were then used to engraft adult Rag2±/± mouse
bone marrow, resulting in the re-establishment of an immune
system (Rideout et al., 2002). Differentiation of these genetically
modi®ed ES cells into fully functional haematopoietic stem/
progenitor cells for bone marrow transplantation has proved
dif®cult, however. Part of the problem encountered is a result of
the Rag2±/± mouse having active natural killer (NK) cells, and this
results in death of the donor cells. In addition, the bone marrow
cells that engrafted were of the myeloid lineage, not the lymphoid
lineage. The low levels of immunohistocompatibility complexes
on the ES cells resulted in depletion of these cells by NK cells,
and this effect was only prevented by removal of NK cell
function. This report proved that therapeutic cloning combined
with gene therapy was capable of restoring normal immune
function, but also illustrated the absolute requirement for correct
levels and matching of the major histocompatibility proteins for
clinically useful tissue therapeutics.
Adult stem cells
An alternative source of stem cells can be found in adult-derived
tissue-speci®c stem cells. For example, having a reliable source of
blood stem cells from bone marrow, peripheral or UCB abrogates
the need for deriving blood from an ES cell source. This is not the
case, however, for neural and some other tissue-speci®c stem
cells, which are not readily available for collection. Although
neural stem cells have the capability to grow in culture and
repopulate the murine or rat brain, there is no simple way to
obtain brain or other neural tissue for the derivation of stem cells.
Therefore, the derivation of neural and other speci®c stem cells
from ES cells might be a viable alternative.
Bone marrow transplantation
The existence of adult tissue-speci®c stem cells is best
characterized by the haematopoietic system. In the ®rst
documentation of the self-renewal of blood in adults (Till and
McCulloch, 1980), the turnover rate of blood cells was seen to be
great, requiring the system to provide replacement cells throughout the life of the organism. Bone marrow transplantation has
been used successfully to treat haematological disorders,
congenital immunode®ciency diseases and other immunological
failures (Thomas, 1999). It is, therefore, a life-saving procedure
for the treatment of haematological diseases, including cancers.
Haematopoietic stem cells (thought to be CD34+ cells) can be
obtained from the bone marrow and from peripheral blood, with
the principal source of haematopoietic stem/progenitor cells at
present being donor bone marrow. Once a perfect HLA match is
found, bone marrow from the donor must be harvested under
general anaesthesia. The requirement for an identical HLA match
gives the best chance for engrafting and reduces the likelihood of
graft-versus-host disease (GVHD)Ða rejection syndrome which,
in its most severe form, can be life-threatening itself
(Ratanatharathorn et al., 2001). In Canada, the Canadian Bone
Marrow Registry and linked international registries maintain lists
of HLA-typed potential bone marrow donors. Although the
Canadian Bone Marrow Registry is able to match the majority of
Caucasian Canadians, there is a very reduced match rate for
ethnic and racial minorities (Beatty et al., 1995).
Umbilical cord blood HSC transplantation
UCB has been established as a clinical source of HSC, which
were ®rst used for a successful bone marrow transplant in a
patient with Fanconi's anaemia in 1988 (Gluckman et al., 1989).
Both in utero and at birth, HSC are found in the fetal circulation,
but within hours after delivery they migrate to the bone marrow
where they provide the progenitors of all blood-forming elements,
including erythrocytes, leukocytes and platelets. In addition to the
fetal circulation, HSC are also found in the 100 ml or so of blood
in the placenta and umbilical cord, which are typically discarded
after delivery. The UCB is easily collected at birth, with no risk to
either mother or baby. The HSC contained in the UCB are
naturally a perfect HLA match for the donor, and also have a high
likelihood of being a perfect or very close match for siblings and
other relatives.
UCB offers many advantages over bone marrow and
peripheral blood-derived stem cells. These include a high
concentration of HSC, less HLA restriction for donors, lower
risk of viral contamination of the graft, a reduced risk of GVHD
(Rocha et al., 2000), and non-invasive collection (Rubinstein et
al., 1998). However, the number of HSC available from a single
cord is limited and, at present, the yield is usually suf®cient only
to engraft children and small adults (Rogers et al., 2001). With
improved collection techniques and more ef®cient cell enrichment
methods, the yield of stem cells is rising and may lead to
successful engrafting of adults as well as children.
At the present time, over 1000 cord blood transplantations have
been performed world-wide to treat patients with malignant and
non-malignant diseases (Ballen et al., 2001). Most of these have
been from sibling donors with partial or complete HLA matching.
Engrafting success has been in the range of 81 to 85%,
demonstrating equivalent success to bone marrow (Barker et al.,
2001; Gluckman et al., 2001). The high rate of engrafting was
associated with a cell dose of 1.53107 nucleated cells per kg body
weight of the patient (Ballen et al., 2001). In addition, recent data
demonstrated that the use of UCB stem cells for transplantation
resulted in reconstitution of a completely normal immune system
despite the very immature and naõÈve nature of the transplanted T
cells (Talvensaari et al., 2002). The incidence of acute GVHD has
been in the range of 10 to 12%Ðonly about half of the expected
rate in a paediatric population receiving bone marrow transplants.
27
I.Rogers and R.F.Casper
Interestingly, analysis of data from North American and European
cord blood transplant programmes did not show any association
between acute GVHD and HLA match, suggesting that the use of
cord blood itself is responsible for the reduced risk (Gluckman et
al., 2001; Yu et al., 2001). As a result of tolerance of cord blood
transplants to more HLA mismatches, fewer cord blood stem cell
donors should be required to meet world-wide needs than with
bone marrow. In addition, and in contrast to bone marrow
transplants where general anaesthesia and surgical transfer of the
donor marrow to the recipient is needed, UCB stem cell
transplantation involves simple intravenous infusion of the
HSC, which ®nd their way to the bone marrow for engrafting.
Case report
The ®rst cord blood transplant from the Toronto Cord Blood
Programme was performed in 1999, in a 34-month-old male with
beta-thalassaemia major. The patient was severely affected,
required multiple blood transfusions, and was at risk of
developing haemochromatosis. The patient's height was 85.5
cm, weight 12.2 kg, and body surface area 0.54 m2. A cousin was
born and had cord blood collected with the HSC cryopreserved;
the cousin's cord blood sample contained 1.543108 nucleated
cells/kg and was a perfect 6/6 HLA match, making it an excellent
donor sample. The patient's bone marrow was conditioned with
chemotherapy, after which the donor HSC were thawed and
infused intravenously into the patient. At 3 weeks post transplant,
the patient received his last platelet and blood transfusion. His
platelet count (mm3) at 4 weeks was 20 000, at 5 weeks was
50 000, and reached 100 000 at 6 weeks. The patient's absolute
neutrophil count was 1000 at 2±3 weeks post transplant. No
signi®cant complications were experienced following the transplant, and the boy was tapered off cyclosporin by 6 months post
transplant. The recipient's ABO blood group was originally O+,
but by 3 months post transplant his new blood group was
con®rmed to be B+ (donor type). Since this ®rst case of HSC
transplantation in the programme, three other samples have been
utilized to treat haematological malignancies, including one
autologous transplant of cord blood HSC, all of which have been
successful.
Haematopoietic stem cell expansion
There are two types of repopulating haematopoietic cell: (i)
progenitor cells, which have limited renewal capacity; and (ii)
stem cells, which are contained within the progenitor population
and have a much greater capacity for self-renewal (Bhatia et al.,
1997). Importantly, some culture conditions that result in an
increase in the number of stem/progenitor cells as measured by invitro methods, for example colony-forming units (CFU) or longterm cell-initiating culture (LTC-IC), do not result in a
proportional increase in long-term engrafting cell numbers
(Piacibello et al., 1999; Dorrell et al., 2000). Increasing the
number of stem cells available for a transplant is critical for
broadening the uses of these cells. The pool of stem cells is small,
and a steady state seems to be maintained throughout most of the
life of the organism, although a stem cell can divide and produce
two new stem cells, two differentiating cells, or one of each
(Abkowitz et al., 2000; Muller-Sieburg et al., 2002). The key
barrier to in-vitro cell expansion, therefore, is to reduce the loss of
self-renewing stem cells that occurs during induced cell
28
proliferation. In order for one stem cell to give rise to two new
stem cells, it is necessary to block the differentiation pathway by
triggering proliferation prior to the onset of the cell's internal
differentiation programme. Most attempts to cause HSC to
proliferate leads to differentiation, since growth factors have
both mitogenic and differentiation properties (Huber et al., 1998).
It is believed that ES cell lines, established in both murine and
human species, can provide a good model on which to base adult
stem cell research. The study of ES cells can provide insight into
controlling the proliferation and differentiation of HSC or other
adult-derived stem cells.
Mesenchymal and blood stem cells
A fascinating aspect of the study of adult stem cells is the discovery
of their multipotency. These cells, much like ES cells, have the
ability to give rise to tissues which differ from the stem cell of
origin. Some of the best examples are blood to brain and skin to
neural transitions (Bjornson et al., 1999). One of the ®rst cell types
to show characteristics of multipotent adult-derived stem cells
were the mesenchymal progenitor cells (MPC) from the bone
marrow stromal cell population. These cells were initially overlooked because they were thought to comprise a mixture of diverse
cell types, which accounted for their ability to give rise to the many
different cell types in culture. This supposition has now been
disproved, and identi®cation of individual cells from the bone
marrow that are capable of diverse tissue development illustrates
the incredible multipotential of these cells (Jiang et al., 2002).
The bone marrow consists of haematopoietic cells and stromal
cells, both of which are capable of multilineage, non-blood
differentiation. A population of bone marrow stromal cells was
identi®ed that was initially quiescent but could be induced to
divide 20±25 times without differentiation. These stromal cells
were positive for a-smooth muscle actin, SH2, SH3, SH4
(mesenchymal cell markers), von Willebrand factor (vWF;
endothelial cell) and extracellular matrix (ECM) proteins
(®bronectin). The range of cells that can be produced from the
bone marrow stromal cell population is extensive (Conget and
Minguell, 1999; Minguell et al., 2000). Although they show
plasticity within the mesenchymal cell types, the ability to
develop into non-mesenchymal cells demonstrates their full
potential as stem cells. Studies have demonstrated the ability of
bone marrow-derived mesenchymal cells to develop into neural
(Eglitis and Mezey, 1997; Mezey et al., 2000), muscle (Wakitani
et al., 1995; Prockop, 1997) and liver (Theise et al., 2000a,b)
tissues.
Tissue therapeutic studies of bone marrow stem cells
The ability of a clonal population of murine mesenchymal
progenitor cells to contribute to multiple tissue types has recently
been illustrated (Jiang et al., 2002). Cells isolated from the bone
marrow appear to have similar properties to ES cells. Single
mesenchymal stem cells have the ability to undergo 120 divisions
and give rise to cell types of all three germ layers (endoderm,
mesoderm and ectoderm), and require LIF for their survival.
These cells also express the ES cell markers OCT-4, Rex-1 and
SSEA-1. Injection of murine-derived bone marrow stromal cells
into the lateral ventricles of newborn mice populated many areas
of the brain and differentiated into glial ®brillary acidic protein
Stem cells: you can't tell a cell by its cover
(GFAP) or neuro®lament-positive cells (Kopen et al., 1999).
Human bone marrow stromal cells show similar properties.
Isolated human mesenchymal cells were induced to undergo
neuronal differentiation in vitro, expressing neuronal-speci®c
enolase (NSE) and tubulin. These cells followed a normal
differentiation pathway, initially expressing nestin (a marker of
neuronal precursors) within 5 h of induction, followed by a
decrease in nestin and an increase in NSE levels (Woodbury et al.,
2000).
Bone marrow stem cells also have the ability to produce liver
cells, and reports exist of murine bone marrow-derived cells
repairing hepatic injury (Theise et al., 2000a,b). To assess
whether or not this property is contained within human cells, a
retrospective study was performed in bone marrow transplant
recipients where there was discordance of donor±recipient gender.
A combination of sex chromosome-speci®c ¯uorescence in-situ
hybridization (FISH) coupled with immunohistochemistry for
liver-speci®c markers clearly showed that bone marrow cells
contributed to functional liver cells within the recipient (Theise et
al., 2000a,b). The recipients received high doses of radiation in
order to deplete their bone marrow of cells, and most likely
received some hepatic injury at the time of bone marrow
engrafting. It is important to note that the stromal and
haematopoietic cells of the bone marrow were not separated in
the above experiments, so it not possible to determine which cell
type contained the stem cells that differentiated into hepatic cells.
It is likely that both stromal and blood haematopoietic cells can
give rise to ES-like cell effects. HSC puri®ed from bone marrow
and transplanted into recipient mice were capable of differentiation into hepatocytes (Lagasse et al., 2000) and of rescuing a liver
defect. In another study, HSC were puri®ed and clonal
populations were examined to determine their self-renewal and
differentiation potential. Long-term repopulation (LTR) of
irradiated hosts was used to show that these cells migrated to
the bone marrow, but could also differentiate into epithelial cells
of the liver, lung, gastrointestinal tract and skin (Krause et al.,
2001).
The contribution and long-term engrafting of many different
tissues demonstrates the potential of bone marrow cells in tissue
therapeutics.
Mechanism of adult stem cell transdifferentiation
It is not surprising that ES cells are multipotent due to their
source, but it is surprising that adult stem cells have the ability to
be multipotential. It is assumed that the lineage restriction which
cells develop protects the organism from unauthorized tissue
development. Evolution may have developed a redundant system
where restriction of competent cell fate occurs due to regulation
by surrounding signalling cells. The generalized potential
manifested by adult stem cells is fascinating, but caution must
be shown in determining a possible mechanism. Recent evidence
that donor cells may fuse with surrounding cells and adopt their
fate suggests that, although the stem cell is contributing to the
repair of the tissue, the mechanism may not be by transdifferentiation but rather by fusion (Terada et al., 2002; Wurmser and
Gage et al., 2002; Ying et al., 2002). Another study (Clarke et al.,
2000) demonstrated the expanded potential of neural stem cells by
co-culture with ES cells. The success of the experiment was
dependent on the ES cells having direct contact with the neural
cells. In light of recent reports on fusion, the multi-tissue potential
of neural stem cells may be a result of fusion of these cells with
the ES cells. In a subsequent study, neural stem cells co-cultured
with C2C12 cells (a muscle cell line) resulted in the conversion of
the neural stem cells to muscle, but contact with the C2C12 cells
was required. Clusters of neural cells separated from C2C12 cells
within the culture failed to generate muscle characteristics.
Moreover, once the neural stem cells had been induced to
differentiate towards mature muscle cells they lost their ability to
differentiate back into neural cells (Galli et al., 2000). At the
present time, therefore, one possible explanation for the
transdifferentiation of adult stem cells into various tissues may
be fusion of the adult cells with stem cells.
Another possible mechanism is that of cell signal transduction.
Although the development of the embryo to an adult follows a
linear process of differentiation, this does not mean that the cells
have a restricted potential. Correct embryo development is
dependent on spatial and temporal differentiation cues, suggesting
that in many cases the cells respond to instructive signals. Studies
conducted in Xenopus embryos have indicated that a window of
time occurs in which individual cells can respond to speci®c
signals. The competency of a cell to respond to inductive signals
is fairly ¯exible when cells are tested in a disaggregation or cell
transplantation system. In contrast, lineage-marking studies,
which retain the cells in situ, indicate a more limited potential
for the blastomeres (Gimlich and Gerhart, 1984; Dale and Slack,
1987; Kageura, 1990; Kimelman et al., 1992; Vodicka and
Gerhart, 1995; Heasman, 1997; Horb and Slack, 2001).
Cell marking studies in mouse embryos (Lawson et al., 1991)
have shown that epiblast cells of the early streak are limited in
potency, and that cells will reproducibly contribute to speci®c
tissues, such that a mouse fate map can be made. In contrast, cell
transplantation studies have shown that the cells take on the fate
of the host tissue (Parameswaran and Tam, 1995). Studies using
late streak cells illustrate that the totipotency is lost with time and
with ingression through the primitive streak. This paradox is
explained by the fact that it is not a result of a cell autonomous
predetermination, but the timing of the passage of the pluripotent
cell through the primitive streak where they are exposed to signals
from surrounding cells (Tam and Beddington, 1987; Lawson et
al., 1991).
A similar situation may occur with adult stem cells. It is
important to determine the developmental limits of these cells.
For example, a haematopoietic stem cell in the bone marrow will
receive lineage-restricted signals so that it develops into a blood
cell; however, placing it in the liver allows it to receive liverspeci®c signals and alter its differentiation programme so that it
becomes a functional hepatocyte, as observed in human bone
marrow transplant patients (Theise et al., 2000a,b).
The question remains as to whether adult stem cells truly are a
`blank slate' ready to respond to any signal or whether they are
limited progenitor cells that need to fuse with a more embryonic
cell in order to transdifferentiate. Likewise, it is unclear whether
there is a rare stem cell in adult tissues that is similar to an ES
cell, or whether adult stem cells are capable of being
reprogrammed (Figure 1). In-vitro studies must be carefully
interpreted because the presence of cell-speci®c markers does not
guarantee functionality, which is the important end point in these
29
I.Rogers and R.F.Casper
studies. In-vivo studies, utilizing clonal cell populations or single
cells strongly suggest the presence of a multipotent stem cell. It is
important to verify the ability of adult-derived stem cells to
replace damaged or diseased tissue with functional cells. In order
to use these cells safely for tissue therapy, the mechanism(s) of
stem cell transdifferentiation must be elucidated. One possibility
is that stem cells are responding to signals involved in tissue
healing. In the case of HSC transplantation, the recipient mice are
irradiated, and this results in tissue damage. The cell signalling
that occurs in the damaged tissue (e.g. liver) and which triggers
the cell repair mechanism may also trigger transdifferentiation of
the HSC. There is evidence that bone marrow cells may be
capable of `curing' Parkinson's disease (Azizi et al., 1998), which
occurs due to the loss of dopamine-expressing neurones due to
apoptosis. It is possible that repair signals from the surrounding
neurones may trigger transdifferentiation of the bone marrow
cells which, after transplantation, ®nd their way to the substantia
nigra and into dopamine-producing neurones (Nagatsu, 2002).
The ability to reprogramme a terminally differentiated adult
somatic cell supports the idea of transdifferentiation of adult stem
cells.
Recently, one group managed to use B-cell nuclei for somatic
nuclear transfer (SNT) into oocytes and for the creation of
functional ES cells (Hochedlinger and Jaenisch, 2002).
Previously, the most widely recognized report of SNT resulting
in the reprogramming of the nuclei in order to generate a live
organism was the cloning of the sheep `Dolly' in 1997 (Wilmut et
al., 1997). Although there is some debate of the true differentiation state of the cell used in the latter study, these two
investigations prove that a terminally differentiated cell can be
reprogrammed to produce multiple cell types.
The reprogramming of nuclei that is observed may be
accounted for by a disruption of normal chromatin states and
methylation patterns of the DNA that come about due to the
exposure of the chromatin to novel cytoplasmic factors of the
recipient cell (Theise and Krause, 2002)
Are umbilical cord blood HSC multipotent?
It is unclear whether other haematopoietic tissues have the same
ability as bone marrow to be the source of multipotential cells. As
mentioned above, human UCB is a source of clinically useful
HSC, supplying enough cells for paediatric bone marrow
transplants. The ability of umbilical cord-derived stem cells to
produce non-haematopoietic cells has not yet been extensively
documented. Haematopoietic mesenchymal cells may be isolated
from the Ficoll layer of UCB and form an adherent population,
thus allowing for their separation from haematopoietic progenitor
cells by differential adherence selection (Erices et al., 2000).
After 3 weeks of growth in serum-containing medium, 25% of the
cultures had mesenchymal cell properties [as de®ned by
morphology and ¯uorescence-activated cell sorting (FACS)
analysis], while 75% developed osteoclast-like properties.
Similar to bone marrow mesenchymal cells, UCB mesenchymal
cells were CD45±, CD34± and did not express the endothelial
marker, CD31. Growth of these cells in osteoblast differentiation
medium (dexamethasone, phosphate and ascorbic acid) or
adipocyte medium resulted in positive identi®cation of osteo-
30
Figure 1. Studies suggesting the existence of a multipotent adult-derived stem
cell have not distinguished between the possibilities that: (i) a tissue-speci®c
stem cell has been reprogrammed; or (ii) a stem cell with embryonic cell
properties is capable of responding to signals to which it is not usually
exposed.
blast-like or lipid-containing cells respectively, thereby con®rming their mesenchyme-like properties.
One group (Mareschi et al., 2001) isolated an adherent cell
population from UCB which, in contrast to the report by others
(Erices et al., 2000), was CD45+, CD34+ and CD31+ but did
contain many osteoclast-like cells. Furthermore, conditions that
encourage the growth of bone marrow mesenchymal cells resulted
in the death of the UCB-adherent cells. Different growth
conditions were used in these two studies, and this may account
for some of the discrepancies in the analysis. The observation that
both blood and mesenchymal cells isolated from the bone marrow
were capable of transdifferentiation, coupled with the possibility
of a mesenchymal-like cell in UCB, merits further investigation
of the potential of UCB as a source of multipotential stem cells.
In preliminary experiments in our laboratory, human UCB
CD34+ cells were tested for non-blood gene expression using
PCR. Interestingly, cells grown in 10% serum showed a
mesenchymal cell morphology and were positive by PCR for
bone (translocon-associated protein; TRAP), muscle (desmin),
neural (nestin), and astrocyte (GFAP) markers (Figure 2). The
numbers of positive cells remained low, but these results
con®rmed previous ®ndings (Erices et al., 2000) and demonstrated the potential of UCB cells to undergo transdifferentiation.
It will be important to isolate the stem cell within the UCB that
is capable of transdifferentiation, and to identify growth
conditions that will promote its proliferation while maintaining
the cells' ability to be multipotential. In addition, having enough
cells to make clinical therapeutics possible represents be a major
hurdle that must be overcome. This is similar to the cell expansion
problem encountered with the use of HSC for bone marrow
transplantation (Madlambayan et al., 2001). The HSC expansion
studies will serve as a paradigm for the expansion of multipotential progenitor cells from UCB.
Stem cells: you can't tell a cell by its cover
Figure 2. (A) Umbilical cord blood cells were grown in serum-containing
cultures and produced mixed cultures of adherent and non-adherent cells. (B)
These cultures contained translocon-associated protein (TRAP)-positive cells,
indicating the presence of osteoclasts. (C) Cultured cells were also positive by
PCR analysis for GFAP (upper panel), nestin (middle panel) and desmin
(lower panel).
Conclusion
Stem cells represent a complex cell type that is not easily de®ned.
The derivation of ES cells has allowed the development of models
of differentiation that have assisted our understanding of embryo
development and tissue formation. The therapeutic use of ES cells
is a clear goal, but for human ES cells this is highly controversial.
To some extent, the properties of ES cells are found in adult stem
cells. We believe that the establishment of human ES cell lines for
research purposes is important in providing a model from which
to extend the studies of adult-derived stem cells. For example, it
will be fascinating to determine if adult stem cells have the ability
to acquire the same embryonic markers in culture as ES cells. The
goal for this research should be the establishment of adult stem
cells as a source for tissue therapeutics, thereby obviating the
need for the use of human ES cells in the future.
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