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Blood, Vol. 94 No. 7 (October 1), 1999:
pp. 2179-2191
REVIEW ARTICLE
By
From the Department of Surgery and the Center for Fetal Diagnosis and
Therapy, Children's Hospital of Philadelphia, University of
Pennsylvania, Philadelphia, PA; and the Department of Medicine,
Veterans Administration Medical Center, University of Nevada, Reno, NV.
IN UTERO HEMATOPOIETIC stem cell
transplantation (IUHSCTx) is a theoretical alternative to postnatal
stem cell transplantation (SCT) for the treatment of congenital
hematologic disorders that can be cured by SCT, and can be diagnosed
early in gestation. Advances in prenatal diagnosis and molecular
analysis now allow diagnosis of the majority of congenital hematologic
disorders by10 to 12 weeks' gestation. The evolution of
high-resolution ultrasound and precise interventional techniques have
solved the technical obstacles to performing early gestational cellular
transplants. Considering these advances, it stands to reason that there
is increasing clinical interest in performing IUHSCTx and that there will inevitably be an increasing number of attempts to treat fetuses with hematologic disorders in utero.
The rationale for consideration of IUHSCTx is based on normal
developmental ontogeny. The early gestational fetus is immunologically immature and uniquely tolerant to foreign antigen, allowing acceptance of allogeneic or xenogeneic cells without the need for
immunosuppression. Under specific circumstances, the fetal environment
appears permissive to engraftment of HSC without the requirement for
myeloablation. The maternal womb is the ideal sterile isolette,
allowing the potential for immunologic reconstitution before birth.
Finally, successful prenatal transplantation could preempt clinical
manifestations of the disease, avoiding the need for postnatal
treatment and the high cost in human suffering, and expense to society,
currently associated with SCT.1,2 This is the clinical
promise of IUHSCTx. To date, this promise remains unfulfilled. With
increasing experimental and clinical experience, the naive concept that
a simple transplant in utero might cure a large number of diseases has
given way to a realistic appreciation of the obstacles to successful
engraftment. Reality has forced reconsideration of the original
assumptions about fetal transplant biology, and resulted in formulation
of new questions. It has also resulted in the consideration of new strategic approaches for the therapeutic application of IUHSCTx, in a
variety of clinical circumstances.
The best supporting evidence that IUHSCTx might work remains an
"experiment of nature" first described by Owen in
1945.3 He observed that dizygotic cattle twins that share
cross-placental circulation were born chimeric for their siblings'
blood elements. This state of "mixed chimerism" persists for life
and is associated with specific transplantation
tolerance.4,5 Natural chimerism has been observed in other
species as well, most notably, humans6,7 and the cotton-top
tamarin (primate).8,9 Interestingly, it has been observed
that donor hematopoiesis in some chimeric animals can actually
predominate, with the persistence of very high levels of donor-derived
cells. This experiment of nature represents "proof in principle"
that, under specific circumstances, allogeneic donor cells can
competitively populate a hematopoietically normal recipient, with
substantial and stable levels of donor cell expression.
Efforts to reproduce "natural" chimerism in the laboratory by the
prenatal transplantation of allogeneic or xenogeneic HSC have had
variable degrees of success. The most successful animal model remains
the sheep. Early gestational transplantation of allogeneic, fetal
liver-derived HSC into normal sheep fetuses results in a high rate of
sustained multilineage hematopoietic chimerism10 that
persists for many years and is typically in the range of 10% to 15%
bone marrow (BM) and peripheral blood donor cell
expression.11 The fetal sheep model is also permissive for
widely disparate xenogeneic engraftment. Multilineage hematopoietic chimerism has been well documented after human fetal liver-derived HSC
transplantation12 and after transplantation of a variety of
human cord blood and adult BM-derived populations.13-18 In
addition, we have shown that chimerism in the human sheep model is
caused by the engraftment of pluripotent HSC by documentation of
long-term engraftment by donor cells on retransplantation into
second-generation fetal lamb recipients.19 In contrast to
the sheep, however, other normal animal models have shown much greater
resistance to engraftment after in utero transplantation. Although
chimerism has been achieved in the normal primate,20
goat,21 rat,22 and mouse,23-26 the
levels of engraftment are much lower and well below what might be
expected to be therapeutic for most hematologic diseases.
In contrast to normal animal models, it is clear that under
circumstances where there is a competitive advantage for normal cells,
high levels of donor cell engraftment can be expected. This was first
shown by Fleischman and Mintz27 in studies in W mutant anemic mouse strains that have a stem cell deficiency based on
the absence of c-kit. In utero transplantation of normal allogeneic
fetal liver cells by transplacental injection at 11 days gestation
resulted in rescue of severely anemic mice and complete reconstitution
by donor hematopoiesis. The degree of erythroid replacement correlated
with the degree of underlying anemia, with complete early replacement
by donor erythroid cells in the lethally anemic W/W homozygotes, and
partial but progressively increasing replacement by donor erythroid
cells in the sublethally anemic Wv/Wv
homozygotes. Donor white blood cell (WBC) engraftment was also seen in
the Wv/Wv recipients, but was not as extensive
as erythroid engraftment, mirroring the underlying severity of the
lineage defect. In a less severe model of anemia based on a different
mutation of c-kit (W41/W41),28 Blazar et al29
documented high levels of multilineage chimerism of congenic donor
cells with confirmation of HSC engraftment by repopulation of
irradiated secondary recipients. Similarly, in the mouse severe
combined immunodeficiency disease (SCID) model in which there is early
arrest of T- and B-cell development, Blazar et al30 have
demonstrated lymphoid reconstitution after IUHSCTx. In successfully
reconstituted animals, T and B lymphocytes were entirely of donor
origin. Although donor myeloid and erythroid elements could not be
consistently detected, the engraftment of donor HSC in the marrow was
clearly documented by retransplantation experiments. Thus, in the
presence of a lineage deficiency, IUHSCTx can selectively reconstitute
the defective lineage, but it appears that competitive pressure from
the normal host lineages prevents high-level multilineage donor cell
expression. More recently, engraftment was compared in the SCID mouse
model following IUHSCTx versus nonconditioned postnatal
SCT.31 There were a number of advantages favoring IUHSCTx
found in this study, including a lower risk of graft-versus-host
disease (GVHD) and more rapid and earlier lymphoid reconstitution of
the thymic and splenic compartments. Recent studies in the nonobese
diabetic (NOD)/SCID mouse confirm and expand upon these
observations.32 In this model, the defect in T- and B-cell
development is the same as the SCID mouse but in addition there are
known defects in natural killer (NK) cells and antigen
presentation.33 IUHSCTx in NOD/SCID recipients results in
multilineage engraftment with increasing donor cell expression over time.
There have now been 26 human cases of IUHSCTx that have been reported
in the literature or are personally documented by the authors. There
have also been a significant number of attempts that are either pending
or have not been reported that cannot be commented upon in this review.
Transplants have been performed by numerous investigators, for many
different diseases, using a variety of transplant protocols. The
authors have recently reviewed the reported clinical experience with
IUHSCTx2 and this is updated in
Table 1. It is important to note that of
the 20 non-SCID cases, 12 have been performed beyond 14 weeks'
gestation, when the human fetus would be expected to be
immunocompetent. In addition there have been at least 3 early deaths, 1 from sepsis (fetal liver source) and 2 procedural that should, in the
modern era, be avoidable. Two cases (1 SCID and 1
The primary determinant for expanding the clinical application of
IUHSCTx will be the ability to improve engraftment in the hematopoietically competitive recipient. To improve engraftment in
competitive prenatal environments, the unique transplantation biology
of the prenatal recipient must be better defined and the barriers to
engraftment identified. IUHSCTx differs in 3 major respects from
postnatal SCT. First, there is abundant evidence that the myeloablative
regimens and irradiation used to permit engraftment after postnatal SCT
alters the biology of the recipient hematopoietic
microenvironment.48-53 Second, after IUHSCTx, there is
competition from a preexisting, vigorous, host hematopoietic compartment that is not present after myeloablation. Third, there is
the underlying framework of normal hematopoietic and immunologic ontogeny. Therefore, the paradigm of postnatal SCT does not necessarily apply to IUHSCTx. Insight into the barriers to prenatal engraftment can
only be obtained by consideration of biologically relevant competitive
model systems. Figure 1
schematically depicts the models and contrasts the
competitive circumstances for donor cells. The following discussion
summarizes what, in the authors' view, is the current experimental
evidence derived from these models that is directly relevant to
IUHSCTx. This evidence will be presented in the context of 3 assumptions that have been used in the past as a presumptive basis for
IUHSCTx.
Assumption no. 1: There is "space" in the expanding fetal
hematopoietic compartment that is available for homing and engraftment
of donor cells.
During fetal development, there is sequential migration of
hematopoiesis from the yolk sac and/or para-aortic splanchnopleure to
the fetal liver and, subsequently, to the BM.54,55 There is
an associated exponential expansion of the hematopoietic compartment with presumably continuous formation of new microenvironmental sites,
or "niches," for homing and engraftment of circulating HSC. We
and others have suggested that the number of "niches" available
for engraftment in the prenatal microenvironment probably exceeds the
availability of niches in the postnatal environment, offering one
explanation for the ability to engraft fetal recipients without
myeloablation. That precept has been challenged by a number of recent
prenatal and postnatal observations. The dogma that the creation of
"space" is required for the engraftment of donor cells after
birth has been increasingly challenged, primarily on the basis of data
derived from the syngeneic nonmyeloablated mouse model, originally
described by Brecher et al56 and recently revitalized by
Stewart et al.57 In this model system, analogous to
IUHSCTx, there is no irradiation effect and the host hematopoietic compartment is intact. However, it differs from allogeneic IUHSCTx in
that engraftment occurs in the postnatal BM environment and donor and
recipient cells are genetically equal and syngeneic with stromal elements.
Assumption no. 2: Donor HSC can effectively compete with host
HSC to achieve significant donor cell expression after IUHSCTx.
By this assumption, successful reconstitution is dependent on the
ability of an initial inoculum of donor HSC to survive and expand into
the host hematopoietic space. If donor cells have a competitive
advantage, then even the engraftment of a relatively limited number of
donor HSC could ultimately reconstitute the recipient. In
normal animal models, we have observed after IUHSCTx little evidence
that donor cells can expand their presence in the host milieu, except
in the human/sheep model when a competitive advantage is conferred by
infusion of donor species-specific human cytokines.12,71 In
contrast, there is abundant evidence that in circumstances of donor
cell competitive advantage, donor cells rapidly expand into a deficient
compartment. The high levels of donor hematopoiesis achieved in
c-kit-deficient mouse strains in which there is a proliferative defect
in host HSC support this hypothesis. Mintz et al72 have
documented full reconstitution in this model after IUHSCTx by 1 or 2 normal HSC. In a separate study, Fleischman73 showed that
when donor cells have equivalent c-kit function to host cells, W mutant
mice do not accept grafts more readily than wild-type animals,
supporting a competitive advantage, rather than space, as the primary
determinant of donor cell expression.
Hypothesis no. 3: The early gestational fetus is immunologically
tolerant of foreign antigen.
Since Billingham et al's82 classic observations of
"acquired" immunologic tolerance, the phenomenon of fetal
tolerance has been relatively accepted. Evidence is now overwhelming
that the fetal thymic microenvironment plays a primary role in
determination of self recognition and repertoire of response to foreign
antigen. Pre-T cells undergo positive and negative selection during a
series of maturational steps in the fetal thymus that are controlled by
thymic stromal cells.83,84 The end result is deletion of T-cell clones with high affinity for self antigen in association with
self-MHC, and preservation of a T-cell repertoire against foreign
antigen. Therefore, theoretically at least, introduction of foreign
antigen before thymic processing should result in presentation of donor
antigen in the thymus with clonal deletion of alloreactive T cells.
However, it is important to note that the mechanism of central thymic
tolerance has been defined primarily in T-cell receptor
(TCR) transgenic mice. In these mice, thymic maturation of lymphocytes
occurs in an environment of unregulated high levels of TCR with high
affinity for a specific self antigen, which is expressed from the
earliest to the latest stages of thymic development.85-87 This is distinct from the clinical situation after IUHSCTx in which
there are a large number of circulating antigens interacting with
recipient TCRs of varying affinity for donor antigen. Differences in
thymic maturation of lymphocytes in normal mice from the defined mechanisms in TCR transgenic mice have been
recognized.88,89 In addition, there are other mechanisms of
rejection including NK- or B-cell-mediated response that are
relatively poorly understood. In fact, experimental efforts to induce
tolerance by prenatal presentation of antigen have had inconsistent
results. In the initial report of Billingham et al,82 only
3 of 5 survivors were tolerant of donor skin grafts (in an MHC class I
disparate but class II matched strain combination), and in many other
investigations, particularly in xenogeneic combinations, results have
been inconsistent.90-92 These classical studies are
difficult to interpret because no analysis of donor cell chimerism
could be performed. In more recent studies, we failed to demonstrate
specific tolerance induction for allogeneic renal grafts in recipient
lambs made chimeric by in utero transplantation of T-cell-depleted
adult marrow, despite the measured presence of 2% to 5% donor
hematopoietic engraftment.93 Carrier et al24
documented specific tolerance to skin grafts in only 3 of 22 mice with
microchimerism after fully allogeneic IUHSCTx. In this same
microchimeric model we have shown that tolerant animals exhibit a
combination of partial clonal deletion and clonal anergy of residual
donor reactive cells, whereas in nontolerant animals no evidence of
deletional tolerance is present.94 In general, these
studies support the existence of the phenomenon of fetal tolerance but
suggest that it may be conditional and dependent on timing and
appropriate presentation of antigen in the fetus.
Consideration of IUHSCTx in the context of the above discussion
suggests a number of strategies by which higher levels of engraftment
in competitive systems might be achieved. These strategies fall into
the category of either increasing the number of donor HSC engrafted or
increasing the competitive capacity of donor-derived hematopoiesis
relative to host hematopoiesis. Strategies designed to increase the
number of donor HSC engrafted must assume a model of at least equal
competitive capacity of the donor cells after engraftment. In this
model, the level of engraftment would be equal to the fractional
representation of donor HSC in the host environment. By this model, it
is clear that far higher numbers of HSC would need to be engrafted than
are currently engrafted after IUHSCTx. Direct approaches of increasing
the number of donor HSC by increasing cell number or HSC enrichment
have been tested to a limited extent in animal models without dramatic
increases in engraftment. However, it is fair to say that the upper
limits of this strategy have not been explored in fetal models, and if host cells can be "displaced" by massive doses of donor
cells,59 such a strategy might be successful. The clinical
limitation to this strategy when using adult BM as a donor cell source
is the T-cell dose administered with higher numbers of
CD34+ cells.47 Another direct approach is to
perform multiple transplants in hopes of maintaining circulating levels
of donor cells to engraft as niches form or become available. This
strategy has been highly successful in the sheep with significant
increases in engraftment even when the same absolute numbers of cells
are given.46 Multiple prenatal transplants have also been
given clinically with success in X-SCID,38,39 where there
is a selective advantage for normal cells, but no appreciable
engraftment was achieved using similar protocols in CGD or
Any consideration of fetal therapy must take into consideration
maternal and fetal risk. The risks of IUHSCTx can be divided into
procedural risks and biological risks for the mother and fetus. The
procedural risks are relatively well characterized and can be
extrapolated from extensive obstetric experience with chorionic villus
sampling (CVS), amniocentesis, and fetal transfusion and
blood sampling. The maternal risks from these procedures (ie, infection, hemorrhage, infertility) independent of fetal loss is
negligible. The risk of fetal loss or other fetal complications from
CVS has been well documented and is less than 1%.98 The procedural risk of IUHSCTx before 14 weeks' gestation has been previously analyzed and is probably also less than 1% per
transplant.2 Therefore, using our current protocol of 3 transplants, we would anticipate a procedural fetal loss rate of no
more than 4%. The biologic risks to the mother and fetus include
infection with bacterial, fungal, or viral pathogens from the donor
cells, fetal GVHD, Rh sensitization for future pregnancies (if the
donor cells are Rh-positive and mother and fetus are Rh-negative), and
maternal graft-versus-host phenomenon ("autoimmune" disease) if
donor lymphocytes cross the placental barrier and survive in the
mother.99 Many of these risks can be minimized by using
adult sources of donor cells (rather than fetal liver) with careful
screening for infectious disease, and scrupulous T-cell depletion. We
currently limit the T-cell dose to less than 1 × 105
CD3+ cells/kg estimated fetal weight as a precaution
against GVHD. The risk of Rh sensitization can be avoided by the use of
Rh-negative donor cells, if possible; if not, sensitization can be
prevented by administration of Rh-immune globulin. The risk of donor
cells crossing the placenta and surviving in the maternal circulation is probably remote, but no data exist. An important concern is whether
IUHSCTx would in any way prohibit what is considered current optimal
standard of care for a given disease after birth. At the present time
there is no rationale to expect that it will, and in fact there is good
reason to think that it can potentially facilitate postnatal SCT if
tolerance is achieved.
It is clear from the above discussion that there are a large number of
diseases that might be considered as targets for IUHSCTx. However, it
is also clear that each disease must be considered individually and may
or may not have favorable enough biology or an adequate rationale for
attempting IUHSCTx. Table 2
categorizes selected candidate diseases by rationale for
IUHSCTx. In contrast to a decade ago, there is now
adequate clinical and experimental information available to guide
rational clinical application of this approach.
Although in the absence of a selective advantage only low-level
chimerism can be reasonably expected after IUHSCTx, low-level chimerism may carry with it the tremendous advantage of donor-specific transplantation tolerance. This would have the clinical effect of
providing a donor without antigenicity after birth. As discussed above,
in the absence of immune response, there is increasing evidence that
engraftment can be achieved with minimally myeloablative strategies.
Particularly for diseases that have been shown to be treatable by
stable mixed chimerism, postnatal "booster" transplants could be
performed to augment the minimal chimerism achieved in utero with
relatively minimal toxicity (Fig 3).
|
TOP
INTRODUCTION
"NATURAL" HEMATOPOIETIC...
-thalassemia)
underwent umbilical blood sampling at 24 weeks, and termination was
performed when no peripheral donor cell expression was
found.
X174, which requires intact T-cell help for B-cell Ig class switching (unpublished data, June 1999). In other immunodeficiency disorders,
such as chronic granulomatous disease (CGD) (3 cases)
-thalassemia attempted, of
which 2 were intrauterine deaths.
chain
(X-SCID), or components of it's signaling pathway (ie, Jak 3 or
ZAP-70).