(Journal of Leukocyte Biology. 2003;73:547-555.)
© 2003
by Society for Leukocyte Biology
Hematopoietic stem cells: can old cells learn new tricks?
Anthony D. Ho and
Michael Punzel
Department of Medicine V, University of Heidelberg, Germany
Correspondence: Anthony D. Ho, Department of Medicine V, University of Heidelberg, Hospitalstr. 3, Heidelberg, Baden-Württemberg, 69115, Germany. E-mail: anthony_dick.ho{at}urz.uni-heidelberg.de
ABSTRACT
Since the establishment of cell lines derived from human embryonic
stem (ES) cells, it has been speculated that out of such "raw
material," we could some day produce all sorts of replacement
parts for the human body. Human pluripotent stem cells can be
isolated from embryonic, fetal, or adult tissues. Enormous self-renewal
capacity and developmental potential are the characteristics
of ES cells. Somatic stem cells, especially those derived from
hematopoietic tissues, have also been reported to exhibit developmental
potential heretofore not considered possible. The initial evidences
for the plasticity potential of somatic stem cells were so encouraging
that the opponents of ES cell research used them as arguments
for restricting ES cell research. In the past months, however,
critical issues have been raised challenging the validity and
the interpretation of the initial data. Whereas hematopoietic
stem-cell therapy has been a clinical reality for almost 40
years, there is still a long way to go in basic research before
novel therapy strategies with stem cells as replacement for
other organ systems can be established. Given the present status,
we should keep all options open for research in ES cells and
adult stem cells to appreciate the complexity of their differentiation
pathways and the relative merits of various types of stem cells
for regenerative medicine.
Key Words: plasticity potential • transdifferentiation • microenvironment • division history
INTRODUCTION
In terms of ontogeny and discovery, hematopoietic stem cells
(HSC) are considered to be "old." In contrast to embryonic stem
(ES) cells, the first stem cells associated with hematopoiesis
appear in the yolk sac on day 10 of gestation in mice and at
6 weeks of gestation in humans [
1
]. In developmental biology,
between a zygote—the ultimate totipotent stem cell—and
specific organs with terminally differentiated cells, there
are numerous stem cells in various stages of development with
more limited self-renewal capacity and developmental potential.
Initially, stem cells that are totipotent give rise to those
that are pluripotent, from which precursor cells for specific
organs and tissues are derived. Within this hierarchy in ontogeny,
HSC are considered to be in an "elderly" stage of development
and hence limited in their developmental potential. The discovery
of HSC also dates back to 1963, and they were the first "stem
cells" that have been defined as such [
2
3
4
].
IN THE BEGINNING WAS THE HSC
The present enthusiasm and simultaneous controversy around stem-cell
research began with two breakthroughs: successful cloning of
Dolly by Ian Wilmut, Keith Campbell, and co-workers [
5
] and
the establishment of human ES cell lines by James Thomson et
al. [
6
]. Without any doubt, these technologies have opened
up novel avenues for tissue engineering and organ transplantation
in humans [
7
]. Never in the history of biomedical research
have discoveries spawned such tremendous repercussions, not
only in the scientific community but also in the society at
large.
Modern day stem-cell research, however, began much earlier with the discovery of assays to detect HSC by James Till, Ernest McCullough, and Lou Siminovitch in 1963 [2
, 3
]. In a murine model, this group provided evidence for the existence of HSC in the bone marrow (BM). Their series of experiments demonstrated that first of all, cells from the BM could reconstitute hematopoiesis and hence rescue lethally irradiated, recipient animals. Second, by serial transplantations, they have established the self-renewal ability of the original BM cells. When cells from the splenic colonies in the recipients were transplanted into other animals that received a lethal dose of irradiation, colonies of white and red blood corpuscles were again found in the secondary recipients. Based on these experiments, HSC were defined as cells with the abilities of unrestricted self-renewal as well as multilineage differentiation. This discovery marked the beginning of modern day stem-cell research. Only in recent years were other somatic stem cells identified in tissues with a more limited, regenerative capacity [8
, 9
].
The first successful attempts using BM transplantation as a treatment strategy for patients with hereditary immunodeficiency or acute leukemias were performed in the late 1960s/early 1970s [10
11
12
13
]. The original idea was to replace the diseased BM with a healthy one after myeloablation. Without the benefits of present-day knowledge of immunology and supportive care, morbidity and mortality rates associated with the treatment procedure were then high [13
]. Nevertheless, the results were considered encouraging as compared with those obtained with conventional treatment options. BM transplantation has in the meantime been proven to be the only chance of cure for some patients with malignant and hereditary diseases [1
]. Its success was a result of the presence of HSC in the marrow graft, which were able to reconstitute the blood and immune systems after myeloablation. Although initially identified in the marrow, HSC could also be found in the peripheral blood upon stimulation, such as during the recovery phase after myelosuppressive therapy [14
] or after administration of cytokines [15
, 16
]. Such HSC obtained from the peripheral blood or isolated CD34+ cell populations have been used successfully in lieu of BM to reconstitute hematopoietic and immune functions in the recipients [17
, 18
]. Meanwhile, innumerable patients owe their life today to BM transplantation as a treatment strategy. Knowledge gained from blood stem-cell transplantation has also shown that somatic stem cells possess the ability to migrate to sites where they are needed—a phenomenon designated as "homing."
REINVENTING HSC
Paramount to the success of BM transplantation is an adequate
amount of HSC in the graft, autologous or allogeneic. There
have been numerous attempts to expand the HSC population ex
vivo, but progenitors with self-renewal capacity seem to be
very demanding. Reports of successful expansion of HSC derived
from human adults in the laboratory have thus far been controversial
and have yet to be reproduced. In contrast, ES cell lines have
been shown to provide an inexhaustible source of stem cells
for all tissue types, and their self-renewal capacity seems
unlimited [
6
,
19
]. It is conceivable that many erroneous organ
functions can be corrected through transplantation of an appropriate
amount of cultured or "engineered" stem cells. For example,
it has been shown that ES cells could be used to cure myelin
deficiency disease in a murine model of human myelin deficiency
such as multiple sclerosis [
20
]. Analogous treatment approaches
in humans might be promising in the future. ES cells have been
induced to differentiate into neurons in a rodent model, which
might be useful for treatment of Parkinson’s disease [
21
].
A successful attempt with cardiomyocytes derived from ES cells
to replace damaged heart muscles has been reported [
22
]. Another
promising strategy is the use of ES cells that are induced to
differentiate into insulin-forming cells (islet cells of the
pancreas) for treatment of diabetes mellitus [
23
]. A recent
report has demonstrated that it was possible to derive hematopoietic
progenitors from ES cells under specific conditions [
24
]. However,
for various reasons to be discussed later, the feasibility of
using ES cells in a clinical setting has yet to be proven, whereas
adult stem cells have been in clinical use for almost four decades
and have been shown to be safe for donors and recipients.
Parallel to the encouraging developments in ES cell research, a number of studies have reported data indicating a similar plasticity potential of somatic stem cells from an adult animal, especially those derived from hematopoietic tissues. Using in vivo transplantation models in rodents, several investigators have claimed that the developmental potential of the stem cells derived from the BM has been underestimated. Evidence gathered from these experiments challenges the hitherto prevailing dogma that cellular differentiation and lineage commitment are irreversible processes and that conversion of somatic stem cells from one germinal derivation to those of another is not possible.
In one of the first studies [25
], unmanipulated BM cells injected into skeletal muscle, which was chemically induced to undergo regeneration, were found to participate in the muscle regeneration process. Furthermore, BM cells that have engrafted in the muscle were also involved in the repair process if muscle injury was experimentally induced again at a later time. Using BM transplantation in the mdx mouse model of Duchenne’s muscular dystrophy, Bittner et al. [26
] reported that stem cells within the marrow of mice possessed the ability to form differentiated skeletal muscle fibers and that even cardiac muscle cells were able to regenerate by recruiting circulating marrow-derived stem cells. Gussoni et al. [27
] were able to confirm the results obtained with the mdx mouse. In addition, they showed that a novel population of stem cells, the so-called muscle side population (SP), was able to give rise to dystrophin-positive myofibers and muscle satellite cells. In another study performed by Goodell’s group [28
], a population of muscle SP cells was cotransplanted along with BM cells into irradiated murine recipients in a competitive repopulation assay. They showed that the muscle SP cells had a remarkable capacity for hematopoietic differentiation. Subsequent studies have, however, demonstrated that these SP cells actually originated from the BM [29
, 30
].
Eglitis and Mezey [31
] were the first to show that BM cells were able to differentiate into microglia and astroglia cells within the central nervous system in a murine model. In two other separate studies, Brazelton et al. [32
] and Mezey et al. [33
] suggested that BM cells that were transplanted intravascularly into lethally irradiated mice migrated to the brains of the recipients and differentiated into cells expressing neuronal markers. These studies have provided evidence that mesoderm-derived BM cells could adopt neural and hence ectodermic cell fates.
Petersen et al. [34
] showed that stem cells from the rat BM were able to give rise to hepatocytes in recipients pretreated with 2-acetylaminofluorene, a suppressor of hepatic proliferation, and carbon tetrachloride, an inducer of hepatic injury. BM transplantation from a healthy animal was able to rescue the recipients from lethal hepatic failure by transdifferentiation into ductular cells and hepatocytes. In a different study [35
], mice were subjected to whole-body irradiation followed by BM transplantation. The presence of donor-derived, mature hepatocytes was documented in the liver of the recipients, showing that BM-derived stem cells probably participated in hepatocyte restoration.
Whereas stem cells from the marrow with such transdifferentiation potentials have been presumed to be HSC, BM is composed of HSC and a variety of supportive cells collectively referred to as stroma. The latter is probably derived from mesenchymal stem cells (MSC) and represents a niche for the HSC. In vivo and in vitro studies have shown that the marrow-derived MSC were able to differentiate into adipocytes, cartilage, bone, tendon, vascular smooth muscle [36
37
38
], cardiac muscle [39
], and lung [40
]. It remains debatable whether one single-cell type between MSC or HSC is responsible for the plasticity or transdifferentiation potential of BM cells or whether different subsets, each having completely transdifferentiation potentials, are present. Although these issues will probably remain controversial for the next few years, there is nevertheless evidence that pluripotent population(s), if at all, are derived from hematopoietic tissue, i.e., the BM.
In this regard, a more recent study in the murine model by Lagasse et al. [41
] has established that more purified populations of HSC were in fact able to generate hepatocytes in vivo after transplantation. That HSC might rescue animals from lethal hepatic failure, albeit inefficiently, by replacing defective hepatocytic elements was demonstrated by Grompe and co-workers [42
] in a murine model. This group has shown that wild-type-purified HSC from adult donor animals could repopulate and regenerate the liver of lethally irradiated recipients with hereditary tyrosinemia type 1. Krause et al. [43
] reported multiorgan, multilineage engraftment by a single BM-derived stem cell with HSC phenotype. Their data have provided one of the few indications that multiple tissues could develop from a single tissue-derived HSC. The magnitude of engraftment was, however, minuscule such that the biological relevance has been questioned. Weissman and his group [44
] generated chimeric animals by transplantation of a single green fluorescent protein-marked HSC into lethally irradiated, nontransgenic recipients. Single HSC robustly reconstituted peripheral blood leukocytes in these animals, but did not contribute to nonhematopoietic tissues, including brain, kidney, gut, liver, and muscle. Their data indicated that "transdifferentiation" of circulating HSC and/or their progeny is an extremely rare event, if it occurred at all [44
].
A number of reports have indicated that stem cells, especially MSC derived from human BM, might exhibit a similar degree of plasticity [45
46
47
48
49
50
]. Verfaillie and co-workers [51
52
53
54
] recently described the isolation and ex vivo expansion of multipotent adult progenitor cells (MAPC) from adult BM in humans as well as in rodents. These cells were expanded in culture for more than 70 passages without signs of senescence and could differentiate at the single-cell level, not only into MSC but also to cells of visceral mesodermal origin such as endothelium. If confirmed to be reproducible by others, such MAPC lines might indeed represent ideal sources of stem cells for replacement therapy.
We have shown that after liver transplantation, donor-specific CD34+ cells could be detected in the recipient’s BM [55
]. In a recent report, Körbling et al. [56
] provided indications that stem cells derived from peripheral blood could differentiate into mature hepatocytes and epithelial cells of the skin and gastrointestinal tract. Although the magnitude of engraftment is minimal (i.e., at most 7% of hepatocytes were of donor origin), the latter study suggested that generation of donor-derived human hepatocytes after transplantation of HSC from the donor could be a possibility.
The possibility of myocardial repair after infarction by transplantation of HSC has generated enthusiasm worldwide. In a mouse model, Orlic et al. [57
] have shown the regeneration of cardiomyocytes after myocardial infarction by direct administration of purified Lin- c-kit+ BM cells into the healthy myocardium, adjacent to the site of the infarct. After 9 days, regenerating myocardium from the highly enriched HSC was shown in the damaged heart. Jackson et al. [29
] transplanted the so-called SP cells, regarded as highly enriched HSC, into lethally irradiated mice, subsequently rendered ischemic, and could confirm the cardiomyogenic potential of HSC. By means of transplantation of allogeneic HSC, other authors have provided evidence for generation of vessel endothelium and revascularization of an artificially induced myocardial infarction and subsequent regeneration of the infarcted heart muscle [58
]. Albeit these studies provided only weak evidence, and more research is absolutely required, they have nevertheless spawned a series of trials intended to bring this into clinical practice.
PLASTICITY OF ADULT STEM CELLS: ALL HYPE AND NO HOPE?
Adult stem cells from several nonhematopoietic tissues have
also been reported to produce cell types other than those from
the tissue in which they reside. Bjornson et al. [
59
] demonstrated
that neural stem cells could produce a variety of blood cell
types after transplantation into irradiated hosts. Clarke et
al. [
60
] also reported this observation that adult neural stem
cells might have a broader developmental capacity. The latter
showed that neural stem cells from the adult mouse brain could
contribute to the formation of chimeric chick and mouse embryos
and gave rise to cells of all germ layers.
More recent experiments have severely challenged the notion that blood cells could be derived from neural or muscle stem cells. For example, in the experiments described by Bjornson et al. [59
], the cells from neurospheres that were dissociated and transplanted were passaged 12–35 times in the presence of growth factors before transplantation. One possible explanation for the loss of specificity of neural stem cells is that they were transformed during their in vitro passaging. Iscove and his co-workers [61
] repeated the experiments, confirming that transformation of primary neural stem cells occurred during in vitro passaging but could under no circumstances observe any contribution of neural cells to the blood cell lineage. The SP cells, originally identified in murine skeletal muscle, have been shown by subsequent experiments to be derived from the BM and considered as highly enriched HSC [29
, 30
]. Hence, these recent data led to the conclusion that transdifferentiation could not be proven, and validity of many of the plasticity experiments was challenged [62
].
Furthermore, recent studies from Ying et al. and Terada et al. [63
, 64
] have provided evidence that cell fusion between somatic stem cells and ES cells occurred spontaneously upon coculturing in vitro. Both groups cautioned that such hybrid cells with tetraploid nuclei and characteristics of somatic stem cells and ES cells could account for the proclaimed plasticity potentials of somatic stem cells hitherto reported.
Most of the experiments performed thus far have focused on the dramatic changes in the destiny, i.e., differentiation program of adult stem cells. Transdifferentiation or in some rare examples, plasticity seemed indeed possible under highly selective pressure from the microenvironment. There is, however, an absolute paucity of data on the cellular and molecular processes involved in the complex cascade of transdifferentiation. Even the very first step, which is probably communication between somatic stem cells with the surrounding cells in the microenvironment, has not been elucidated at all. Evidences at a cellular and molecular level show that reprogramming along a different differentiation pathway is lacking. It is also not known how the newly acquired differentiation program can be maintained. Before these are known, it is premature to translate the observations in animal models into clinical trials.
LESSONS FROM THE PAST FOR THE FUTURE
From the many in vivo studies indicating the possibility of
transdifferentiation potentials of adult stem cells, the following
principles can be drawn. First, an initial demand for regeneration
of the corresponding cell type must exist before transdifferentiation
can occur. This has been induced by injury inflicted artificially
or by the urge for growth of all organ systems in a fetal environment.
For example, transdifferentiation from HSC to hepatocytes might
be achieved after the recipient animal has been exposed to radiation
or chemical damage to the liver [
41
,
42
,
49
]. Conversion
of donor HSC into cardiomyocytes could occur only after induction
of myocardial infarction [
57
] and into vascular endothelial
cells after ischemia induced through ligation of a coronary
artery [
58
]. Hence, transdifferentiation was observed only
when the need for growth arose. How such demands are defined
and which factors are responsible remain future challenges.
Second, adult stem cells are able to migrate or "home" on their own accord to the tissues where the demand arises, i.e., to the organs injured by chemical or radiation insult. In most of the experiments mentioned, stem cells from a donor animal have been transplanted via a peripheral blood vessel. Direct administrations of HSC into the affected area, e.g., for the regeneration of cardiomyocytes in a rodent infarction model, were exceptions [58
]. The same group of investigators has however demonstrated that in the presence of an acute myocardial infarct, a significant degree of tissue regeneration could be achieved 27 days after mobilization of HSC by a combination of stem-cell factor and granulocyte-colony stimulating factor in the same rodent infarction model [65
]. They concluded that mobilization of HSC from the autologous marrow by cytokines was able to induce the regeneration of myocardium lost. For patients undergoing BM transplantation in a clinical setting, HSC are transplanted by infusion into a central venous access after the recipient has undergone a conditioning regimen, and the HSC migrate and home to the marrow. The biological and molecular mechanisms underlying stem-cell homing have not yet been defined. Signals from the injured tissues and adhesion molecules might play a role. The question of which factors and chemokines stimulate the migratory activity of the stem cells toward the sites of demand has so far evaded elucidation.
Third, once settled in a niche after homing, surrounding cells in the microenvironment play a major role in defining the long-term fate of an adult stem cell. They seem then to be "educated" by the neighboring cells to maintain a balance between self-renewal and differentiation [66
]. Some authors have argued that the specific, supportive cells in the niche are responsible for stem-cell renewal, coupled with opposing signals from others that promote differentiation of daughter cells displaced outside the niche [67
]. Others [68
] indicated that a combination of adhesion molecules and cytokines might accomplish similar effects. Nevertheless, differentiation has been demonstrated to occur along the pathway of the predominant cell type into where they have homed and not along the pathway of the organs of their origin. For hematopoiesis, coculture with a mitotically inactivated layer of marrow stroma cells, the feeder layer, is required for assessment of long-term repopulating activity and for stem-cell self-renewal [69
]. In somatic stem cells, this regulation of self-renewal versus differentiation has been shown to occur extensively in a coordinated manner. In contrast, cells derived from ES cells might exhibit their pluripotency in an uncontrolled manner such that ectopic cell growth, e.g., teratocarcinoma, might arise in the recipient [70
, 71
].
THE IMPORTANCE OF THE STEM-CELL NICHE: IN ROME, DO AS THE ROMANS DO
Therefore, the stem-cell niche provides a means to regulate
stem-cell numbers as well as to control differentiation pathways
in vivo [
72
]. These two fundamental decision processes can
best be appreciated in HSC, as they have been relatively well
characterized and the significance of interactions between HSC
and marrow stroma cells appreciated [
73
,
74
]. Cross-talk between
HSC and marrow stroma cells might serve as an ideal model to
identify the determinants and the crucial signaling pathway
for supporting (trans)differentiation and how the balance between
self-renewal and differentiation is achieved. A thorough understanding
of these fundamental mechanisms is essential to appreciate the
power and the means to manipulate the long-term fate of somatic
stem cells.
HSC are characterized by the dual abilities to self-renew and to differentiate into progenitors of multiple blood cell lineages. These two features require that HSC undergo at some stage asymmetric divisions to generate cells to sustain long-term hematopoiesis as well as the various progeny cells of the distinct blood lineages. The two daughter cells from a HSC might be initially equivalent to replenish the stem-cell pool, but subsequent cell divisions must result in different fates of the progeny cells [75
, 76
]. Alternatively, a balance between symmetrical cell divisions that results in self-renewal versus that which results in differentiation might be able to maintain the stem-cell pool and provide a source of multipotent progenitors. Even if the latter were true, asymmetric division must have occurred during ontogenesis of these two populations.
To follow the precise replication history and the subsequent long-term fate of HSC at a single-cell level, we have applied a time-lapse camera system to directly monitor early cell divisions. Using a combination of technologies for monitoring cell-division history and long-term growth behavior of the same HSC at a single-cell level, we have been able to define the impact of regulatory molecules and cellular elements that determined the symmetry of divisions and long-term fate [77
, 78
]. The following conclusions can be drawn. First, we have definitively confirmed that asymmetric divisions correlate with primitive stem-cell function. CD34+/CD38- cells that have divided asymmetrically gave rise to more blast colonies than those showing symmetric divisions. Cells giving rise to primitive myeloid-lymphoid-initiating cells (ML-IC) were associated with asymmetric divisions and demonstrated significantly lower division kinetics than those giving rise to committed colony-forming cells [78
, 79
]. Second, the percentage of such asymmetric divisions decreased with ontogenic age [77
]. Third, whereas significant changes in mitotic rate, colony formation, and asymmetric divisions were dependent on exposure to regulatory molecules, the asymmetric division index (ADI)—i.e., the ratio of cells undergoing asymmetric divisions versus dividing cells—remained constant [77
]. Therefore, commitment decision to self-renewal versus to differentiation is probably not influenced by regulatory molecules [80
, 81
]. Exposure to the HSC supporting an AFT024-feeder layer was, however, able to elevate the proportion of cells undergoing asymmetric divisions, recruiting more primitive stem cells into cycle and giving rise to more primitive ML-IC [82
]. Thus, although extrinsic signaling can skew the pattern of commitment and differentiation, self-renewal is governed by direct contact with the stem-cell niche.
Other studies have demonstrated that 
10% of pluripotent progenitors undergo asymmetric cell division. [83
]. A relationship between cell-division rate and asymmetric cell divisions has also been reported, which showed that the proliferative potential and cell-cycle properties were unevenly distributed among daughter cells derived from single-sorted HSC from fetal liver [84
]. These studies also confirmed that expansion potential is associated with asymmetric division, and lineage commitment was not influenced by cytokines [85
].
Two mechanisms may be responsible for the adoption of different fates by the daughter cells. One, the intracellular or intrinsic mechanism, involves an inherited determinant that is asymmetrically segregated into one daughter cell at the time of division [86
87
88
]. The other, an extracellular or extrinsic mechanism, may result from communication of the daughter cells with each other or with surrounding cells [86
]. Recent genetic analysis has identified several proteins that differentially segregate during division and may be involved in determining the asymmetry of division. These determinants range from transcription factors (such as PROS) [89
] to modulations of cell–cell interactions (such as Numb and Notch) [87
] and are asymmetrically localized during division of Drosophila neuroblasts [87
88
89
90
].
Recently Kiger et al. [91
] and Tulina and Matunis [92
] have defined the molecular nature and spatial organization of the signaling pathway that govern stem-cell fate in the Drosophila testis. In the latter, germ line and somatic stem cells attach to a cluster of support cells called the hub. Upon division of a germ-line stem cell, the daughter cell in direct contact with the hub retains the self-renewal potential, whereas the other daughter cell was destined to differentiate into a goniolblast and subsequently into spermatogonia. Both reports provide evidence that Unpaired, a ligand activating the Janus tyrosine kinase (JAK)–signal transducer and activator of transcription (STAT) signaling cascade, expressed by the apical hub cells in the testis, causes stem cells to retain self-renewal potential. Analogous to this finding, the maintenance of mammalian ES cells has been shown to require a similar JAK–STAT signaling, which is counterbalanced by the requirement for mitogen-activated protein kinase activation, and the latter in turn promotes ES cell differentiation [93
].
Our observation that direct communication between HSC with the stroma [82
] is required for maintaining and expanding the self-renewal potential of HSC is compatible with these findings. Further research is necessary to define how the functional cross-talks between HSC and the niche occur, how this education will define the differentiation pathway, and how the balance between self-renewal and differentiation is regulated.
CLINICAL RELEVANCE OF IN VIVO MODELS FOR TRANSDIFFERENTIATION
Most of the experiments demonstrating the transdifferentiation
potential of hematopoietic tissue-derived stem cells have been
performed in rodent models. As findings in mice and rats cannot
always be extrapolated to reflect the situation in humans, studies
evaluating defined populations of human cells in clinically
relevant animal models are needed to evaluate the full potential
of human stem-cell plasticity.
Xenogeneic transplantation models have been developed in immunodeficient mice and preimmune sheep that served as surrogate assays for the in vivo engraftment and differentiation potentials of human HSC [1
, 94
]. The sheep model of human hematopoiesis was developed by taking advantage of the permissive environment of the preimmune sheep fetus. In this model, populations of putative human HSC are transplanted intraperitoneally into early gestational sheep fetuses. In contrast to other systems, this model has a normal functioning immune system but is able to support the engraftment of human HSC. This allows the sheep model to be used for studies into the mechanisms of HSC homing and engraftment, as the recipient’s hematopoietic microenvironment is intact. Long-term engraftment and multilineage expression of human HSC were also accomplished without the use of human cytokines [94
95
96
97
98
99
100
101
].
Using the fetal sheep model, Almeida-Porada et al. [102
] were able to demonstrate that different populations of highly purified human HSC derived from adult BM and mobilized peripheral blood were able to give rise to mesenchymal cells after transplantation, providing evidence that this model system is capable of permitting the transplanted cells to switch from one mesodermic fate to another, namely HSC into MSC. Zanjani et al. [103
] provided evidence that highly enriched populations of human adult BM and umbilical cord blood (UCB) HSC gave rise to functional human hepatocytes within the developing fetal sheep liver. This transdifferentiation from mesodermal HSC to endodermal hepatocytes seemed to occur in the absence of any injury or external stimulus, thus suggesting that stem-cell plasticity is possible under normal, physiologic conditions during fetal development.
Based on these evidences, we propose that this human/sheep in utero transplantation model probably represents an ideal method to evaluate the plasticity potential of human stem cells. In the growing fetus, all the organs have started to differentiate, but the need for exponential growth and differentiation could still permit reprogramming of cellular fate through a bombardment of differentiative stimuli. In this model, transplantation of human cells is performed during the fetal period in which all of the organs are rapidly proliferating and differentiating. The transplanted cells should thus be provided with the opportunity to find the appropriate niche in each organ, assuming that the transplanted cells harbor that potential.
Only through an understanding of the molecular mechanism governing symmetry of division shall we be able to manipulate stem-cell proliferation versus differentiation and hence the long-term fate of stem cells. Our own in vitro data at a single-cell level indicated that stem cells, which have divided slowly and asymmetrically, are associated with a higher self-renewal potential than cells that have divided rapidly and symmetrically [77
, 78
, 82
]. Further studies have suggested that communication with the microenvironment in turn determines the self-renewal and differentiation programs [79
, 82
]. Among epithelial cells, interaction between cells is mediated through formation of junctional complexes. Data from the literature and our preliminary experiments with HSC also support this notion and suggest that a specific differentiation program might be switched on, after direct contact with the surrounding (unpublished results). The human/sheep in utero transplantation model represents an in vivo model to validate all these observations and hypotheses deduced from experiments with human HSC at a single-cell level.
RELATIVE MERITS OF ADULT AND ES CELLS
The concentration of somatic stem cells in an adult organism,
as well as their self-renewal potential, has been shown to be
extremely limited. The BM, for example, probably contains a
broad spectrum of stem cells at different stages of development.
HSC constitute a small minority of the BM cells and stem cells
with pluripotence, most probably, even far less. HSC that might
produce various blood cell types are likely to be found in the
CD34
+/CD38
- subset, and they represent only 0.001–0.01%
of the BM mononuclear population [
104
]. MAPC, which probably
give rise to MSC and originate from the marrow, are present
in 1–1 million [
51
]. Despite attempts to multiply HSC
from adult BM or UCB, there is thus far no evidence that this
can be accomplished. Long-term repopulating HSC cannot be expanded
in vitro without losing developmental potential [
1
]. Verfaillie
and her group [
51
,
52
,
55
] have however recently demonstrated
that MAPC could proliferate without obvious senescence under
clinically applicable conditions. However, these data need to
be confirmed and validated by other groups.
In contrast, ES cells can readily be expanded without any obvious limitations in culture. Another advantage of ES cells is their ability to form virtually any cell type in the living organism. Whereas many studies, as mentioned above, have demonstrated that adult stem cells, especially HSC, MSC, or MAPC, are able to form a wide variety of cell types under specific conditions, i.e., are able to transdifferentiate, there is no evidence that they can make the whole range of cell types, i.e., possess plasticity potential to a relevant extent.
However, the use of ES cells for transplantation is also associated with hazards. In animal studies, immature embryonic cells might induce teratoma or teratocarcinoma after transplantation into a recipient animal [70
]. Murine ES cells have been shown to be epigenetically instable, and animals formed completely from ES cells seldom survive [71
, 105
]. Preculturing of immature ES cells in conditions that induce differentiation along a specific pathway might reduce this risk of tumorigenesis. ES derivatives were also ineffective at repopulating hematopoiesis in lethally irradiated, adult animals [105
, 106
]. The latter studies indicated that an adult recipient might reject donor cells derived from ES cells. Animal studies have shown that an adult animal would accept only donor ES cells, after a specific differentiation stage. Hence, ES cells must be primed toward a predefined, differentiation pathway before transplantation [20
21
22
, 107
108
109
]. Such cultures are likely to contain a variety of cells in different stages of development as well as quiescent ES cells. Purification of the cell preparation is necessary before clinical use could be considered. By transfecting yolk sac or ES cells with HoxB4, a homesotic selector gene implicated in self-renewal of definitive HSC, Kyba et al. [110
] have demonstrated that primitive HSC can be promoted to become definitive HSC.
All the aforementioned evidence indicated that adult HSC in culture lose their long-term, repopulating potential, and ES derivatives do not repopulate the marrow of an adult in vivo. These observations suggest that in vitro culturing probably shuts down genes that are vital to in vivo maintenance of the stem-cell pool. The observation that HoxB4 expression in ES cells was required for engraftment and hematopoietic differentiation supports this notion. Research in ES cell differentiation can therefore greatly enhance and facilitate our acquisition of knowledge in manipulating the long-term fate of stem cells.
Whereas human ES cells are yet far from any clinical applications, adult stem cells have been in clinical use for almost 40 years, and extensively, studies have demonstrated that they can save lives of patients with leukemia, marrow failure, or hereditary diseases. Above all, stem cells from hematopoietic tissues, i.e., the BM, are readily accessible, and clinical trials have demonstrated that they can be harvested and transplanted safely in an allogeneic or autologous setting.
Fetal tissues could also be a potential source of pluripotent stem cells. Our group has demonstrated a significantly higher proliferative and self-renewal potential among stem cells derived from fetal tissues as compared with UCB or adult BM [104
]. Whether this impressive, self-renewal potential is also associated with higher plasticity potential is currently being studied. Clinical trials with fetal neurons as a treatment approach for patients with Parkinson’s disease or Chorea Huntington have already been reported, albeit with moderate success [111
, 112
]. The acquisition of viable stem cells from fetal tissues for clinical application remains a challenge. Legal abortions are usually performed under specific medical or psychosocial indications and within a narrow window between 9 and 15 weeks of gestation. Samples obtained might show genetic malformation or embryopathy. The material obtained might also be severely damaged or contaminated by infectious microorganisms and not always suitable for therapeutic purposes.
CONCLUSIONS
Stem-cell-based approaches to tissue reconstruction might open
unpredicted opportunities. Despite all the recent criticisms
and doubts cast on some of the earlier experiments, hence challenging
the plasticity potential of adult stem cells, some of the evidence
for transdifferentiation of specific subpopulations has been
intriguing, and this warrants rigorous research to verify this
possibility. Whereas most of the studies focused on the dramatic
change in long-term fate, e.g., conversion into tissues of another
germinal derivation, little is known about the mechanisms of
the initial steps leading to another differentiation pathway,
about the hierarchy of molecular changes involved in switching
to another program, and about the stability of these alterations
in the progeny cells. Cross-talk with the microenvironment probably
determines the long-term fate in terms of differentiation pathway
as well as of the balance between self-renewal and differentiation.
Identification of the differences, at a molecular level in the
interactions between specific subsets of marrow-derived stem
cells with various feeder layers, might be essential to precisely
monitor the mechanisms regulating the long-term destiny of stem
cells. Through an understanding of how the microenvironment
communicates with HSC, we might gain the power to alter their
long-term fate for therapeutic purposes. Eventually, molecular
signals triggered by adhesion and junctional complexes are responsible
for the ultimate expression of specific signaling and differentiation
pathways. Insight into the developmental pathways of various
organ systems from ES cells would also facilitate an understanding
of how adult stem cells can be steered to other differentiation
pathways than were originally destined. Given the present status
in research, we should keep all options open for studies of
ES and adult stem cells to appreciate the complexity of their
differentiation pathways, the power to manipulate a cell’s
destiny, and the relative merits of various types of stem cells
for regenerative medicine.
Received September 13, 2002;
accepted February 3, 2003.
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