Science, 25. Feb 2000, Vol 287, No. 5457, page 1442
Translating Stem and Progenitor Cell Biology to the Clinic: Barriers and Opportunities
Stem cells are the natural units of embryonic generation, and also
adult regeneration, of a variety of tissues. Recently, the list of tissues that use the
model of differentiation from stem to progenitor to mature cell has increased from blood
to include a variety of tissues, including both central and peripheral nervous systems and
skeletal muscle; it is also possible that all organs -and tissues are derived from, and
still contain, stem cells. Because the number and activities of stem cells and their
progeny are homeostatically regulated, clinical stem cell transplantation could greatly
add to the physician's armamentarium against degenerative diseases.
Irving L. Weissman
The clinical application of stem and progenitor cell transplantation began with the
exposure of civilian populations to lethal doses of radiation in 1945. Irradiation of mice
replicated the syndrome, and bone marrow (BM) transplants radioprotected them by providing
donor-derived hematopoiesis (1-3). In 1961, Till and McCulloch demonstrated the existence
of clonogenic BM precursors that give rise to multilineage hematopoietic colonies in the
spleen [colony-forming units, spleen (CFU-S)]; a subset of spleen colonies contained cells
capable of forming more spleen colonies. They proposed that these were pluripotent
hematopoietic stem cells (HSCs) (4-6) that had the property, at the single-cell level, of
(i) self-renewal as well as (ii) multilineage differentiation. This remains the enduring
definition of stem cells.
Whereas the above-described experiments provided evidence that stem cells exist, they did
not enable their isolation. With the development of quantitative assays for clonogenic
precursors in mice of all hematolymphoid precursors (7-9), a reductionist approach was
developed for the identification and isolation of HSCs. Monoclonal antibodies (mAb) were
identified that bind cell surface markers on some, but not all hematopoietic cells; marker
positive and negative subsets were separated by cell sorting (e.g., fluorescence-activated
cell sorting) to identify cells with clonogenic precursor activity (9). Eventually,
clonogenic multipotent progenitors with a distinctive marker profile proved to be HSCs
(Fig. 1A) (10-13). A subset of this population perpetually self renews; these are
long-term stem cells (LT-HSCs) (12, 14). All subsets of these HSCs were radioprotective,
and HSCs were the only radioprotective elements in mouse bone marrow (11). As the HSC cell
dose increased, the time to engraftment of clinically protective numbers of donor-derived
blood cells shortened (Fig. 113) (15). Subsequently, in vitro and in vivo assays for
clonogenic human stem and progenitor hematolymphoid cells were developed and by the same
approach candidate human HSCs were isolated (16, 17).
The mouse and human HSCs depicted in Fig. 1A were the first isolated by surface markers.
It was subsequently shown that both CD34+ and very rare CD34-
subsets of LT-HSCs exist (18, 19); HSCs actively extrude dyes such as Hoechst 33324 and
Rhodamine 123, and can be isolated by this property (20). In humans, the mAb AC133 also
identifies HSC (21).
Purified human HSCs are capable of hematopoietic reconstitution in patients receiving bone
marrow ablative (myeloablative) doses of radiation and chemotherapy. Increasing the dose
of HSC shortens the time to engraftment of mature blood elements in man as in mice (Fig.
1C) (22-24).
Biology of Hematopoietic Stem and Progenitor Cells
In mice, LT-HSCs give rise to short-term HSCs (ST-HSCs), which give rise to
multipotent progenitors (MPPs), whose further progeny are oligolineage-restricted (Fig. 2)
(12); dedifferentiation cannot be detected (25). HSCs can first be found in the developing
yolk sac blood islands; transfer of blood island cells to same age-hosts resulted in
lifelong, donor-derived hematopoiesis (26). HSCs can also be found in the embryo proper
(27, 28). HSC are next found in the fetal liver (13), and then the fetal spleen and BM
(29); each stage occurs presumably by HSCs entering the fetal circulation. In young adult
mice, about 8% of the LT-HSC population randomly enters cell division per day, and on
average, half their progeny must be LT-HSCs to maintain the steady-state level. As HSCs
progress to MPPs, the frequency of cells in cycle increases (30, 31). In very old mice,
most LT-HSCs are in cycle (32).
Dividing HSCs have four developmental choices: self-renewal, differentiation, programmed
cell death, and emigration (33). The frequency of HSCs in hematopoietic organs is
regulated by the fraction of stem cells that choose one or another of these fates.
Transgenic expression of the anti-programmed cell death gene bcl-2 (a proto-oncogene) in
HSC results in an increase in their frequency in BM (34). These HSC have increased chemo-
and radiotherapy resistance, a property that would be much valued clinically if bcl-2
expression could be regulated. The movement of stem cells between primary hematopoietic
sites occurs naturally throughout life (35). Clinical provision of cytokines such as G-CSF
alone or along with cytoreductive drugs [for review, see (36)] can induce mobilization of
stem cells to blood (MPB), where they are collected for transplantation. Natural and
induced HSC mobilization begins with mitotic expansion of HSC, followed by the release of
G1 HSC to blood to seed secondary sites (35).
Oligopotent progenitors downstream of HSCs have also been isolated (Fig. 2) (37, 38); HSC
give rise alternatively to the clonal common lymphocyte progenitor (CLP), or the
clonogenic common myeloid progenitor (CMP). CMP, in turn, can give rise to megakaryocyte
or erythrocyte progenitors (MEPs), or granulocyte/monocyte progenitors (GMPs). None of
these progenitors dedifferentiate or show self-renewal capacity (37, 38).
Broadening the Stem and Progenitor Cell Concept to Other Tissues
In vertebrates, the zygote is a totipotent stem cell, as are virtually all of its
progeny around the blastula stage; cells contained within the inner cell mass (ICM),
include (and may be composed of) totipotent stem cells (TSCs) (Fig. 3) (39). Embryonic
stem (ES) cells are derived from cultures of ICM cells, and have the property of
participating as totipotent cells when placed into host blastocysts. The developmental
pathways that endogenous ICM cells or transferred ES cells take to tissue formation and
organogenesis has led many to hope that these pathways can be controlled for the
development of tissue and organ specific stem cells (40). However, we currently have an
insufficient understanding of the developmental events that lead to organogenesis from ICM
cells to program the production of tissue- or organ-specific stem cells.
During vertebrate development, at defined stages the derivatives of the embryonic germ
layers of endoderm, ectoderm, and mesoderm are involved in tissue formation and
organogenesis. What is not yet clear is whether every tissue uses the stem and progenitor
model shown to be operative in hematopoiesis (Fig. 3). It is reasonable to propose that
most, if not all tissue and organ systems are based on a stem and progenitor model during
organogenesis and that stem cells are retained throughout life to participate in
regeneration and repair. If this thesis is correct, it would follow that the lessons
learned from regeneration and repair of the hematopoietic system might be useful for the
regeneration and repair of other organ systems.
The value of using the body's own stem and progenitor cell plan of tissue and organ
regeneration is that their numbers and fates are regulated. For example, one cannot
deliver too many HSC; regeneration derived from these stem cells results in regulated
hematopoiesis. The advantages of a medicine based on stem or progenitor cell
transplantation are (i) that one need not understand the process in detail to apply the
therapy, (ii) that, the applied therapy should have attendant toxicities only during the
acute phase of host preparation for stem or progenitor transplants, and (iii) that the
therapy is applied just once. In contrast, medical therapies based on substances that
affect endogenous molecular targets will usually have effects and toxicities wherever the
molecule is expressed; such therapies are by their nature chronic and are required for the
duration of the disease.
Rat neural crest stem cells have also been isolated (41). Using as an assay the clonogenic
reconstitution of in vitro multilineage neural cultures, we have enriched for candidate
human fetal brain CNS stem cells (CNS-SCs) (42). The existence of CNS-SCs had been shown
by retrovirus marking of cells (43). Transplantation of clonally-marked cells gave rise to
neurons and glia whose cell fates were dictated by the regional CNS microenvironment (44).
Continuing neurogenesis can occur in the adult brain in particular microenvironments such
as the dentate gyrus and the subventricular zone (45). Candidate CNS-SCs at the
single-cell level can produce neurospheres of multiple neural cell types; expanded numbers
of cells in neurospheres can be prospectively isolated and are clonogenic precursors of
neurospheres (42). These neurosphere cells can be transplanted into immunodeficient
newborn mice or immunosuppressed adult rats and participate in neurogenesis of neurons and
glia.
In skeletal myogenesis, current leading stem cell candidates are the satellite cells (46).
Enrichment of precursors for blood vessels (47) and for skin (48) has been accomplished.
Several unusual outcomes of cell transplantation have been reported: these include blood
derived from clonal neurosphere cultures (49), blood derived from myogenic precursors
(likely satellite cells) (50), myogenesis and vasculogenesis from isolated blood and bone
marrow precursors (51, 52), and even participation of hematopoietic cells in neurogenesis
(53) or liver development (54). It is not clear how this happens. For purposes of this
review, the means by which organ-specific stem cells seem to change fate are relevant only
to the extent that such cells are potential sources of expanding cells for transplantation
(55, 56).
Clinical Transplantation of Stem and Progenitor Cells: Current Practice, Barriers to
Their Accomplishments and Opportunities
Hematopoiesis as a model of stem and progenitor transplantation. BM transplantation
was invented to enable physicians to increase chemotherapy and radiotherapy to
myeloblative doses with the objective of eliminating endogenous cancer cells. The first
transplants that were successful were between identical twins, wherein no
histocompatibility barrier of host against donor, and no opportunity of immune based
reactivity of donor against host, exists (57).
Autologous BM or MPB transplants. Autologous hematopoietic transplants have been used in
many patients with cancers, including those of the hematolymphoid system (lymphomas and
leukemias), of plasma cells (multiple myeloma), and breast cancer (58). But even if these
tumors are sensitive to chemotherapy, only a fraction of patients are cured. Why is this?
First, in many patients, disease recurs at the primary site; thus, in many patients the
level of therapy did not eliminate endogenous tumor. Second, the bone marrow and the MPB
of patients with these cancers are often contaminated with cancer cells (59). Without
elimination of these potentially clonogenic cancer cells, it stands to reason that the
benefits of high dose chemotherapy could be countermanded by the reintroduction of
malignant cells to the circulation. Isolation of human CD34+Thy+ HSC
from MPB can result in the elimination of detectable malignant multiple myeloma (23, 60),
breast cancer (22), and lymphoma cells from the transplant (24). In the trial in Fig. 1C,
the number of transplanted malignant cells was undetectable; further clinical trials with
human HSC and MPB seem warranted. (I would like to warn the reader that I was co-founder
of the company (SyStemix, Inc.) that initiated and carried out the trials, and therefore I
might have biases]. It is likely that widespread cures of malignant disease by HSC
transplants will not occur unless patients are subscribed earlier in the course of their
disease, or if therapies adjuvant to the transplants are attempted. One direction of
adjuvant therapy that can eventually be applied will be to attempt to regenerate or
reconstitute specific immune responsivity to the small amount of residual tumor. For many
tumors, immunity can be induced and is mainly T cell-based. T cell immunity cap detect
tumor-unique antigens or tumor-associated peptides that are derived from proteins specific
to differentiating cells and are presented on the cell surface by self human lymphocyte
antigen (self HLA) molecules (61). The antigen specific T cell receptors that recognize,
for example, HLA-A2 and the enclosed melanoma peptide MAGE are entities that retain their
specificity no matter whose T cell expresses them, opening the possibility of T cell
receptor (TCR) gene transfection to endow antitumor immunity. The collection of T cells
that recognize a particular HLA plus tumor peptide can be detected and isolated by a new
technology of producing fluorescent major histocompatibility complex, (MHC) peptide
tetramers (62). Perhaps TCR transfection. of HSC/CLP/T cells and/or tetramer-based T cell
isolation will enable transplantation of the specific component of immune reconstitution
in patients with minimal residual disease following HSC transplant. Additional strategies
to augment these immune cell therapies include vaccination with gene-altered tumor cells
(63), or augmenting and prolonging the antitumor T cell response by preventing their
shutdown (64).
Allogenic Transplantation of Hematopoietic Cells
Allogeneic hematopoietic grafts are potentially useful in cancer treatment, as they
are not contaminated with cancer cells; unfortunately, BM and MPB contain T lymphocytes
(58, 65). These donor T cells encounter and respond to host antigens in virtually all
tissues in the body, leading to a multisystern graft-versus-host (GvH) syndrome (58).
HLA-mismatched hematopoietic grafts are usually rejected (66). The high degree of HLA
polymorphism makes a random match between unrelated humans a rare event (58). The
probability of an HLA match is 25% between siblings. Because MHC molecules process and
present any of a number of peptides present within a cell, siblings that share HLA may not
share all tissue-specific peptides; these peptides create minor histocompatibility
antigens when presented by shared HLA molecules. Minor histocompatibility antigens are
important for both host rejection of grafts and GvH immunity (67). HLA-matched
host-versus-graft and GvH immunity can largely be controlled by highly immunosuppressive
treatments that have attendant risks of chronic immunosuppression (68, 69). Patients given
limiting numbers of hematopoietic cells often fail to engraft if T cells are eliminated,
but engraft (and get GvH disease) if donor T cells are retained (65). These T cells are
said to -"facilitate" engraftment (70-73). The presence of facilitator cells
raises the hone that one can cotransplant these cells with HSC to facilitate engraftment
without GvH (70-73). However, in mouse models simply raising the HSC dose is sufficient to
guarantee rapid and sustained engraftment in the absence of either failure to engraft or
GvH, even if the mice are full H-2 mismatches (15, 74). For patients without cancer that
require allogeneic hematopoietic or HSC transplants, it would appear that HSC alone at
high doses would be most useful. In mouse studies, HSC doses sufficient to obtain rapid
engraftment in the autologous setting are also doses sufficient to provide engraftment in
the fully allogeneic, MHC mismatched setting (15).
In the case of HLA-matched allotransplants for leukemia, T cells can carry out a
graft-versus-leukemia (GvL) response (75). Contained within the population of GvH T cells
are cells that can-recognize tissue-specific peptides in the context of shared HLA (76).
Clinicians have fine tuned this response to the extent that initial hematopoietic cell
grafts can be followed by donor lymphocyte infusions (DLI), when the patient is much
healthier and when the patient's GvH response has been controlled. A significant fraction
of patients with chronic myelogenous leukemia are in prolonged or complete remission as a
result of DLI (77, 78). Recently, "mini-transplants" of HLA-matched MPB into
sublethally treated hosts receiving drugs more specific for T cell immunity are followed
by DLI; this process provides a lower transplant associated mortality and morbidity,
retaining the benefits of GvL (79).
Allogeneic HSC and progenitor transplants can be used in a nonmalignant setting to restore
the hematolymphoid system of the host (80). For example, a number of monogenetic disorders
lead to deficiencies in cells within the hematolymphoid system, including a variety of
severe combined immunodeficiencies and hemoglobin disorders (65). Rep air of the defective
enzymes or defective globins could come about either by allogeneic HSC transplants, or
autologous gene corrected HSC transplants. The application of transgenic corrections of
hematopoietic stem cells has been slowed by problems at a number of stages, but many of
these problems have been solved [for example, (81)].
The Use of Allogeneic HSCs for the Induction of Specific Lifelong Transplantation
Tolerance
It has been known since the late 1950s that allogeneic bone marrow hematopoietic
grafts into irradiated hosts can lead to donor specific chimerism for the life of the host
[reviewed in (82)]. These hosts have hematolymphoid systems that are derived wholly or in
part from donor stem cells. Such hosts are usually permanently tolerant of donor organ or
tissue transplants. Thus, one can transplant fully allogeneic HSC and cotransplant, for
example, hearts from the HSC donors, and produce specific and lifelong acceptance of the
transplant, with retention of reactivity to third party grafts and pathogens [reviewed in
(80)]. Cotransplantation of HSC and stem cells for other tissues or organs from the. same
donor ought to be possible, and ought to enable a circumstance wherein sublethal
conditioning of the host permits hematolymphoid chimerism for the purpose of tolerance
induction, and cell- and organ-specific regeneration for the replacement of diseased or
destroyed organ systems.
Allogeneic HSC Transplants for MHC-Determined Autoimmunity
Many of the autoimmunities are genetically based, especially those that involve an
autoimmune response of T cells to organ- or tissue-specific antigens, such as in type I
diabetes (the insulin producing islets are their principal target) (83) and multiple
sclerosis (the myelinated nerve sheaths are targets). In these cases, the predilection for
development of autoimmune T cells can map to particular MHC alleles (84). In mice, HSC
transplants from normal donors into lymphoablated diabetogenic (NOD) hosts can abrogate an
ongoing diabetogenic autoimmune T cell response (80). The hosts are tolerant of
subsequently transplanted donor strain islet grafts (85). Thus, allogeneic HSC transplants
can abrogate autoimmunity and induce transplantation tolerance for subsequent stem cell,
tissue, or organ grafts.
Transplantation of Nonhematopoietic Stem Celts
The aforementioned models provide a means by which tolerance can be induced to a
particular donor set of transplantation antigens. In the case of patients with diseases
wherein the generation of mature or maturing cells of a particular organ system is a
central problem, cotransplantation of HSCs and nonhematopoietic stem cells should enable
organ regeneration.
The recent identification of candidate CNS-SCs and the ability to grow them to large
numbers in in vitro cultures should allow testing of the notion that such cells would be
capable of regenerating neural or glial elements when necessary (56). Transplantation of
tissues that include dopaminergic neurons such as adrenal medulla, fetal ventral
mesencephalon, and teratomas are currently being tested in animal models and human cases
of Parkinson's disease (45). Rodent CNS cell lines that include CNS-SC, sometimes
immortalized with v-myc (86), have been used in a number of models of mouse genetic
neurodegenerative diseases, including demyelinating diseases (87), brain gangliosidosis,
and other neurodegenerative disorders (88). It is not clear which is the appropriate cell
to transplant-the CNS-SCs, the required neurons, or the intermediate progenitors between
the two. In the hematopoietic system only HSC are required (11, 15). Although one might
think that in the nervous system more differentiated neurons are the appropriate
transplants, the normal generation and regeneration of different parts of the brain occurs
via stem cells, and it is conceivable that only stem and progenitor cells have both the
migratory capacity and the differentiation pathways capable of treatment of these neural
defects. Thus, in neurodegenerative diseases, it is important first to determine the rules
of transplantation of stem, progenitor, and mature cells, as well as determine the sites
into which the transplants must be placed.
Other potential neurological disease targets include multiple sclerosis, where an ongoing
T cell response might be abrogated by allogeneic HSC transplants or other potent
immunosuppressive maneuvers (89). In these cases, remyelination from endogenous precursors
is not guaranteed, and the use of neurogenic stem cells, their oligopotent progeny, the
immediate precursors of myelinating glia, or the glia cells might provide tissue specific
remyelination and regeneration of function. Other potential targets of neural and
progenitor cell transplants might include tissues damaged by small strokes, spinal cord
injuries, etc.
Transplantation of Other Stem or Progenitor Celts
Liver organ transplants are the therapy of choice in a number of conditions wherein
the liver is damaged by toxins, drugs, viral infection, or if the patients have gene
defects in the production of important liver-generated factors or receptors. For the most
part, liver transplants require a recently deceased but still perfusing donor, and long
waiting lists exist for liver transplants. Of course, because donors who recently died are
most likely not HLA-matched to the recipient, liver transplants are usually HLA-disparate
and require powerful immunosuppression. It is reasonable to assume that if
liver-repopulating stem or progenitor cells are available, sibling transplants may become
feasible.
The identification of islet stem and progenitor cell populations appears to be at an
earlier phase of development (90). Islet cell transplantation would be preferable to
multiple insulin treatments daily, as these are the cells that both. sense the circulating
levels of glucose and respond appropriately by releasing insulin at the right dose and
tempo. The complications of diabetes are frequent, life shortening, and difficult to
manage by insulin therapy. Whole pancreas transplants are difficult, as in liver
transplantation. Islet transplants require large numbers of viable cells, and as yet islet
cells are difficult to expand in vitro. Thus, it is a reasonable goal to search for
conditions wherein islets are continuously generated from stem/progenitor cells, as in
some mouse models (90), and to replicate them in vitro.
Muscle regeneration in the case of the intrinsic muscular dystrophies or muscle loss
conditions could be life-saving. The recent isolation of skeletal muscle satellite stem
cells (46) gives hope that stem cell therapy can be applied to these conditions. Another
frequent target for muscle regeneration is the heart, where rapid cell death following
coronary artery blockage is a major cause of mortality and morbidity. Unfortunately, the
satellite cell equivalent in the heart tissue has not yet been found.
It is reasonable to expect that cotransplantation of HSCs and tissue or organ stem and
progenitor cells will occur increasingly over the next two decades and will result from
the intersecting advances in stem cell biology and stem/tissue transplant immunology.