Biology of Blood and Marrow Transplantation
Volume 14, Issue 7 , Pages 729-740, July 2008

In Utero Hematopoietic Stem Cell Transplantation: Progress toward Clinical Application

  • Demetri Merianos
    • ,
  • Todd Heaton
    • ,
  • Alan W. Flake

      Affiliations

    • Corresponding Author InformationCorrespondence and reprint requests: Alan W. Flake, MD, Department of Surgery Abramson Research Bldg., Rm. 1116B, 3615 Civic Center Blvd., Philadelphia, PA 19104-4318.

Children's Center for Fetal Research, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania

Received 28 January 2008; accepted 15 February 2008. published online 28 April 2008.

Article Outline

Abstract 

In utero hematopoietic stem cell transplantation (IUHCT) is a potential therapeutic alternative to postnatal hematopoietic stem cell transplantation (HSCT) for congenital hematologic disorders that can be diagnosed early in gestation and can be cured by HSCT. The rationale is to take advantage of normal events during hematopoietic and immunologic ontogeny to facilitate allogeneic hematopoietic engraftment. Although the rationale remains compelling, IUHCT has not yet achieved its clinical potential. Achieving therapeutic levels of engraftment by IUHCT alone remains challenging. However, considerable experimental progress has been made toward the clinical strategy of using IUHCT to induce donor-specific tolerance to facilitate a relatively nontoxic postnatal HSCT. Because donor specific tolerance induction requires relatively minimal engraftment, this strategy may hold the key to broad clinical application of IUHCT in the near future.

Key Words: In utero hematopoietic stem cell transplantation, Stem cell, Transplantation

 

Back to Article Outline

Introduction 

Hematopoietic stem cell transplantation (HSCT) represents the only curative therapy for many hematologic disorders. However, in the absence of a matched donor, standard protocols for HSCT entail considerable morbidity and mortality. In utero hematopoietic stem cell transplantation (IUHCT) is a potential nonmyeloablative alternative to HSCT for congenital hematologic disorders that can be diagnosed early in gestation. Through advances in prenatal screening and molecular-based diagnostics, the opportunity for fetal intervention is greater than ever before. The potential advantages of IUHCT over postnatal HSCT are largely based on the immunologic immaturity of the early gestational fetus, providing the opportunity for induction of donor-specific tolerance to allogeneic cells. The phenomenon of fetal tolerance can potentially eliminate the requirement for immunosuppression with its associated morbidity. The potential clinical impact of IUHCT is enormous when we consider the possibility that any disorder that can be prenatally diagnosed, and can be treated by HSCT, might be optimally treated by IUHCT.

Despite the unique opportunities offered by the fetal microenvironment, the clinical promise of IUHCT remains unfulfilled. Levels of engraftment have been well below what might be expected to be therapeutic for most hematologic diseases, and clinical success has been limited to X-linked severe combined immunodeficiency (XSCID). However, experimental and clinical work over the past 2 decades has resulted in a greater understanding of the complexity of the fetal microenvironment and the obstacles it presents to successful engraftment. In this review we will examine the rationale and the experimental and clinical progress that has been made in IUHCT. In addition, we will discuss our view of the primary barriers to engraftment and discuss promising strategies with the potential to overcome them.

Back to Article Outline

Rationale 

The potential advantages of IUHCT are based upon unique events that occur during normal hematologic and immunologic development that favor the successful engraftment of transplanted allogeneic hematopoietic stem cells (HSC). The phenomenon of fetal immunologic tolerance, first introduced by Billingham et al. [1], is perhaps the most important advantage of IUHCT over postnatal SCT. The fetal thymic microenvironment plays a primary role in the determination of self-recognition and the repertoire of response to foreign antigens. Pre-T cells undergo positive and negative selection during a series of maturational steps in the fetal thymus that are controlled by thymic stromal and dendritic cells 2, 3, 4. The end result is the deletion of T cell clones with high affinity for self-antigen, and preservation of a T cell repertoire against foreign antigen. Therefore, introduction of foreign antigen prior to thymic processing should, in theory, result in presentation of donor antigen in the thymus with clonal deletion of alloreactive T cells. Although the concept of fetal tolerance remains valid, it has become apparent that it is not an all-or-none phenomenon. Self-reactive T cells are known to escape thymic deletion in significant numbers and to be controlled by regulatory mechanisms, including T regulatory cell populations, that are essential for prevention of autoimmune disease 5, 6, 7. In the context of IUHCT, this relatively new knowledge has important implications that will be discussed below.

A second potential biologic advantage of IUHCT is that the early gestational period is the only time in life during which the large-scale migration of stem cells to tissue compartments normally occurs. The hematopoietic system is the prototypical example. Hematopoiesis shifts from the yolk sac, placenta, and AGM region, to the fetal liver and finally to the bone marrow 8, 9, 10. Although this was once naively thought to provide “space” in the expanding hematopoietic compartment, it is now apparent that the fetal hematopoietic compartment is highly competitive, containing an excess of circulating HSCs to occupy developing niches. Nevertheless, if one could understand and utilize the natural mechanisms that normally regulate migration and engraftment of native HSC, one might achieve a selective advantage for donor cells. The third major advantage of the fetus compared to the adult is its relatively small size. At 12 weeks' gestation, the human fetus weighs <35 g. Therefore, much higher relative doses of stem cells can be transplanted, favoring successful engraftment. Finally, prenatal SCT offers the potential to preempt early disease manifestations and prevent the end-organ damage and severe treatment-associated morbidity of postnatal HSCT.

Back to Article Outline

Experiments of Nature 

Hematopoietic chimerism between nonidentical, dizygotic twins with shared placental circulation has been observed in multiple species, including cattle, goats, and primates 11, 12, 13, 14. This phenomenon was first described by Owen in 1945 [13] after he observed that dizygotic cattle twins that share crossplacental circulation were born chimeric for their siblings' blood elements. These natural chimeras exhibit specific immunologic tolerance and maintain stable levels of hematopoietic chimerism for life. Specifically, dizygotic twin cattle have been shown to be immunologically tolerant of their sibling by mixed lymphocyte cultures and renal and skin allografts 15, 16. These “experiments of nature” are proof in principle supporting the potential of IUHCT to achieve mixed allogeneic chimerism with associated donor specific tolerance.

Of relevance to clinical application, the existence of chimerism in human 11, 17 and nonhuman primate 14, 18 twins is well documented. In the case of dizygotic human twins, the frequency of chimerism is relatively high (8% for twins and 21% for triplets) [17], and levels of chimerism in some cases have been at a level that would be therapeutic for most hematologic diseases [19]. These findings have long provided proof of principle for the therapeutic potential of IUHCT. However, because natural chimeras result from the mixing of hematopoietic cells via placental vascular anastomoses, the exposure to allogeneic blood components occurs continuously and begins very early in gestation, which is difficult to replicate experimentally.

Back to Article Outline

Experimental Progress—Animals 

IUHCT has been studied in a variety of animal models over the past 30 years. The first experimental success with IUHCT utilized transplacental injection of donor BM cells at E11 into anemic fetal mice with a stem cell deficiency based on the absence of c-kit [20]. In these studies, the degree of erythroid replacement correlated with the degree of underlying anemia, with complete early replacement by donor erythroid cells in lethally anemic homozygous mice. This was the first example of the general principal that IUHCT can be highly successful in model systems or diseases entailing a competitive or survival advantage for donor cells. Later studies by Blazar et al. [21] demonstrated the ability to achieve multilineage chimerism in anemic recipients, as well as the ability to achieve lymphoid reconstitution in the mouse severe combined immunodeficiency (SCID) model 22, 23. Thus, in the presence of a lineage deficiency, IUHCT was able to reconstitute the defective lineage; however, it appeared that competitive pressure from the normal host lineages prevented multilineage donor cell expression. The presence of an immune deficiency also appears to favor engraftment. IUHCT in the nonobese diabetic (NOD)/SCID mouse model, in which there are additional defects in natural killer (NK) cells as well as antigen presentation [24] resulted in multilineage engraftment [25]. These studies demonstrated the advantage conferred to donor cell engraftment after IUHCT by circumstances of competitive advantage, immune deficiency, or both.

Results after allogeneic IUHCT in normal animal models have been more variable. The most encouraging data has been generated in the ovine model, where early gestational transplantation of allogeneic HSCs into normal sheep fetuses results in sustained multilineage hematopoietic chimerism 26, 27. The fetal sheep model is also permissive for xenogeneic engraftment, as persistent, multilineage hematopoietic chimerism has been documented after transplantation of human-derived HSCs 28, 29. Recently, the pig model has also been successfully utilized for IUHCT. Using Swine Lymphocyte Antigen (SLA) mismatched pigs and a technique of T cell depletion and whole BM add back (to achieve 1.5% T cell content) stable and measurable multilineage chimerism was achieved [30] with associated tolerance for SLA donor-matched kidney grafts [31]. Other normal large animal models such as the primate 32, 33, goat [34], and dog [35] have shown much greater resistance to engraftment, with significantly lower levels of chimerism.

Over the past few years, significant experimental progress has been made in the murine model, which was previously highly resistant to engraftment 36, 37, 38. Specifically, mixed hematopoietic chimerism across full MHC barriers with associated donor specific tolerance has been achieved 38, 39, 40. With greater understanding of the requirements for engraftment [41] and the mechanisms of tolerance after IUHCT [42], strategies have been developed that achieve high level or complete donor chimerism across full MHC barriers in the mouse without the need for immunosuppression or myeloablation 43, 44, 45. However, despite recent successes, the efficiency of engraftment in animal model systems remains low and, in mice, somewhat strain dependent, raising the challenge of how to achieve consistent donor cell engraftment after IUHCT.

Back to Article Outline

Barriers to Prenatal Engraftment 

It is obvious from the preceding discussion that, despite the unique opportunities offered by the fetal microenvironment, there are also unique challenges to overcome. The primary issue is that IUHCT, at the present time, must be performed without any myeloablative conditioning. Because of the developmental status of the fetus and the potential repercussions of any pharmacologic toxicity on the fetus and mother, standard conditioning agents cannot be utilized. In the absence of an entirely nontoxic and hematopoietic specific myeloablative approach, success in IUHCT will require novel strategies to selectively engraft donor cells. Although the rationale for IUHCT remains compelling, specific barriers prevent IUHCT from achieving its clinical potential. These barriers can best be understood in the context of 3 broad categories: receptivity of the host hematopoietic compartment, competition from host hematopoietic cells, and immunologic barriers to engraftment [46].

Receptivity of the Host Hematopoietic Compartment 

The concept of receptivity with regard to the host hematopoietic compartment refers to the historic assumption that there is “space” available in the expanding fetal hematopoietic compartment that is available for homing and engraftment of donor cells. However, current evidence disproves this assumption and suggests that the fetus is far less receptive to engraftment of donor cells than postnatal myeloablated or congenic nonmyeloablated recipients. In theory, IUHCT offers the opportunity to engraft cells utilizing the natural mechanisms that normally allow migration and engraftment of native HSC. In the early gestational period, hematopoiesis shifts from the yolk sac, placenta, and AGM region, to the fetal liver and finally to the bone marrow. During this time, there is an exponential expansion of the hematopoietic compartment with presumably continuous formation of new niches for engraftment of circulating HSC. Therefore, it has been assumed that the number of niches available for engraftment in the prenatal microenvironment exceeds the availability of niches in the postnatal environment; however, a number of studies have challenged this assumption.

A relatively valid postnatal model for comparison is the nonmyeloablated syngeneic mouse model. In this model the concept of “space” represents a dynamic equilibrium of stem cell cycling and occupation of niches. Studies in this model, in which the postnatal hematopoietic compartment has not been irradiated and donor and recipient cells are genetically equal, have shown a dose-dependent increase in donor cell engraftment with repetitive large doses of syngeneic donor BM cells 47, 48. These studies suggest that there is a steady state of open receptive sites in normal, nonmyeloablated BM, and that donor cells can displace host cells over time [49]. How does the IUHCT prenatal environment compare with the nonmyeloablated postnatal environment? The only comparable data available is for long-term engraftment, which reflects the relative proportion of host HSC displaced by donor HSC over time. Interestingly, IUHCT of 20 × 106 congenic BM cells [50] results in lower engraftment levels than 40 × 106 syngeneic BM cells in the postnatal nonmyeloablative model [47] (6%-8% versus 11%, respectively). This represents approximately 50 times the cell dose on a per kilogram basis in the fetus arguing against any receptive advantage for the fetus. However, long-term engraftment reflects multiple parameters including homing, engraftment, and subsequent competitive capacity of the donor cells. To assess the early homing and engraftment of donor cells in the fetus we have performed detailed homing studies in the murine model of IUHCT at a developmental time point when the only receptive hematopoietic site is the fetal liver (E14). In 2 separate studies using different routes of administration (intraperitoneal and intravenous) we have documented an engraftment efficiency in the fetal liver for whole BM of <5% 41, 51, and for highly enriched HSC (intravenous administration) of only 0.43% [51].

Logically, the availability of niches for donor cell engraftment in the fetus will ultimately depend on the balance between the expansion of circulating hematopoietic precursors and the formation of new stromal receptive sites. Although Wolf et al. [52] observed that stroma formation precedes hematopoiesis in the fetal liver and BM, he also noted that hematopoietic activity increases very rapidly after the establishment of stroma. A later study demonstrated that the number of HSC and progenitors circulating in fetal peripheral blood is much higher than the number in cord blood, or after birth, supporting the notion of relative HSC excess [53]. In summary, there is little or no evidence that the fetal environment has any significant receptive advantage that facilitates IUHCT.

Competition from Host Hematopoietic Cells 

Unlike the myeloablated postnatal recipient, the fetal recipient after IUHCT maintains a vigorous hematopoietic compartment. Therefore, the success of IUHCT relies on the assumption that donor HSC can effectively compete with host HSC to achieve significant donor cell expression. However, there is abundant evidence that host hematopoietic competition is a formidable barrier to successful engraftment after IUHCT. Experimental evidence supports the notion that when donor cells have a competitive advantage, even the engraftment of a relatively limited number of cells could ultimately reconstitute the recipient. The extreme example of this concept is seen in c-kit-deficient mouse strains that have a proliferative defect in host HSC. In this model, as few as 1 or 2 normal HSC were shown to fully reconstitute the hematopoietic compartment after IUHCT [54]. However, the converse is also true; that is, when the host cells have a proliferative advantage, engrafted donor cells are unlikely to expand. The proliferative and competitive advantages of fetal liver cells 55, 56, 57, 58 and cord blood cells 53, 59, 60 over adult-derived populations are well documented and likely represent a major impediment to expansion of the donor cell compartment when adult donor cells are utilized.

Another relevant observation to IUHCT comes from the allophenic mouse model, in which chimerism is artificially created at the blastocyst level. In this model when donor strains genetically differ with respect to HSC cycling, the more rapidly cycling population will emerge as the dominant hematopoietic contributor 61, 62, 63. These experiments have shown that in a chimeric microenvironment in which allogeneic cells compete, the level of hematopoietic expression is a function of the genetically defined competitive capacity of the HSC.

Another study supporting the overriding importance of competitive capacity when 2 stem cell populations coexist was performed in the previously discussed syngeneic nonmyeloablated mouse model. The model was modified by exposure of the host to minimally myeloablative radiation. This was followed by transplantation of syngeneic donor cells that were either nonirradiated or had received the same dose of radiation as the host [64]. Transplantation of irradiated donor cells reduced engraftment 7-fold, supporting the concept that it is primarily the ability of donor or recipient cells to compete, rather than mere space, which determines successful engraftment. Therefore, it appears that after IUHCT engraftment is limited first by a lack of open receptive sites because of the relative excess of fetal HSC, and subsequently, the ability of the limited number of engrafted donor cells to expand is a function of their competitive capacity relative to the host. Given the rapid cycling and expansion kinetics of fetal HSC relative to adult HSC, it is not surprising that the most common finding after IUHCT of adult BM cells is low-level mixed chimerism at best. An example of the extent of this competition is provided by the murine congenic IUHCT model in which very large doses of donor cells can be delivered directly and reliably into the intravascular system at E14. In this model, 100% of the recipients maintain long-term donor cell multilineage chimerism. However, even with i.v. delivery of massive doses of donor BM cells (2 × 1011 cells/kg fetal wt.), the levels of mixed chimerism are generally well under 10% [50]. Thus, in the absence of any immune considerations or selective advantage for donor cells, host cell competition limits the level, but not the frequency, of chimerism.

The Immune Barrier to IUHCT 

It was stated earlier that fetal immunologic tolerance is perhaps the most important advantage of IUHCT over postnatal SCT. Yet there has been ongoing controversy regarding the significance of an immune barrier to IUHCT. There are a number of indirect arguments supporting an immunologic barrier to engraftment. The fact that the only successful clinical results have been achieved in an immunodeficiency disorder 65, 66, 67, 68 is suggestive, although it is also a disorder that provides a competitive advantage for normal cells. It is also concerning that immunologically active T cell populations in the human fetus have been documented at relatively early gestational time points. First-trimester fetal liver has been shown to contain NK cells as well as T cells that have undergone T cell receptor (TCR) rearrangements and are alloreactive against MHC in vitro 69, 70, 71, 72. In addition, the possibility of a maternal contribution to the immunologic barrier was recently raised when immunologically active cells of maternal origin were tracked to fetal tissues [73]. In a study that may be highly relevant in the context of maternal sensitization to donor cells, maternal autoantibody response has been demonstrated to trigger T cell-mediated neonatal autoimmune disease [74]. Finally, recent studies in which fetal immunosuppression resulted in a 4- to 5-fold increase in chimerism also suggest an immune barrier [75]. Historically, the overriding argument against the presence of an immune barrier was the lack of evidence for an engraftment advantage for congenic over allogeneic cells. In an early study, Fleischman and Mintz [76], using transplacental injection, found increased engraftment for adult BM in MHC matched relative to mismatched c-kit-deficient mice. In this study, however, engraftment could only be achieved in severely anemic mice, and the observation did not apply to fetal liver, which engrafted equally well independent of MHC mismatch. They attributed this difference to developmental acquisition of MHC restriction in adult HSC. In contrast, Howson-Jan et al. [77], using intraperitoneal (i.p.) injection into normal recipients, found a higher incidence of engraftment using allogeneic (5.2%) compared to congenic (0.7%) donors. The results of this study were limited by a low efficiency of engraftment in both groups and the fact that the engraftment was transient. Carrier et al. [36] demonstrated a slightly higher rate of polymerase chain reaction detectable, microchimerism in PB of congenic (25%) versus allogeneic (7%) recipients after IUHCT, but no differences in the frequency of organ engraftment. Recently, using mucopolysacharidosis type VII (MPS-VII) mice, Barker et al. [78] demonstrated persistence of congenic cells using a histologic marker for the enzyme β-glucuronidase (GUSB) in peripheral tissues, but negligible levels of congenic or allogeneic chimerism in the PB. The minimal chimerism and low incidence of engraftment in these studies make interpretation of HSC engraftment difficult. In our own laboratory, with over a decade of experience in the murine model, we, until recently [50], have never documented an advantage for congenic BM engraftment. When considered with the experimental observations cited above, which clearly demonstrated that at least in some circumstances, allogeneic and even xenogeneic cells could engraft, persist, and induce associated donor specific tolerance, the presence of a significant immune barrier seemed unlikely.

However, it is important to note that the mechanism of central thymic tolerance was defined primarily in TCR transgenic mice, in which thymic maturation of lymphocytes occurs in an environment of high-level expression of TCR with high affinity for a specific self-antigen, which is expressed throughout thymic development 79, 80, 81. This is distinct from the microenvironment following IUHCT, in which there is relatively late presentation of allogeneic antigen interacting with recipient TCRs of varying frequency and affinity for donor antigen. Therefore, the question remained as to whether IUHCT could recapitulate immunologic ontogeny. In the murine model we have examined the relationship between engraftment, tolerance, and the presence or absence of clonal deletion utilizing the mammary tumor virus (mtv) oncogenes. When engraftment is successful in the murine model, tolerance is associated with at least partial deletion of donor reactive lymphocytes 39, 42, 43, 44, supporting the concept that for T cell-mediated mechanisms, IUHCT could recapitulate normal mechanisms of self-tolerance.

However, all of these studies were done in the i.p. model, where donor cell doses were limited and there were lingering questions raised by the fact that levels of engraftment in mice were somewhat strain dependent, and by the fact that even when it was clear that equivalent doses of cells were transplanted, only a fraction of recipients engrafted. The question of allogeneic versus congenic engraftment after IUHCT was then reexamined utilizing intravascular (i.v.) injection of donor cells via the vitelline vein, a technique that allows delivery of much higher doses of cells. These studies showed that despite equivalent homing and initial engraftment, by 5 weeks after injection only 30% of allogeneic animals remained chimeric and went on to become long-term chimeras, whereas 100% of congenic animals maintained their engraftment [50], definitively supporting, at least in mice, the presence of an immune barrier to engraftment. The question remains whether this immune barrier is related to some component of innate immunity versus an adaptive immune response. Because all congenic and allogeneic animals maintained measurable levels of chimerism at 2 weeks after IUHCT, it seems that loss of engraftment is a postnatal event, and because the innate immune system should cause earlier loss of allogeneic engraftment, this data supports the existence of a previously unrecognized adaptive immune barrier after IUHCT. The mechanisms involved in this barrier remain to be defined. We have hypothesized, but not yet proved, that donor reactive T cells escape thymic deletion because of inadequate or late presentation of donor antigen in the thymus. These cells either reject the graft or are subdued by a host peripheral regulatory response, analogous to the peripheral regulatory mechanisms that routinely control self-reactive clones that escape thymic deletion in normal individuals. This hypothesis reconciles with our data supporting partial clonal deletion of donor reactive T cells in engrafted animals, and it is consistent with known mechanisms of self-tolerance but requires experimental validation. If true, it would open possibilities for new strategies for facilitation of donor cell engraftment. A major question is how these data relate to a large animal system and the human. By translation of ontologic events the period available for donor antigen presentation in the thymus should be longer. This may provide a better opportunity for donor antigen presentation in the thymus or allow opportunity for manipulation of this process to achieve more complete deletion of donor reactive T lymphocytes or enhance the production of donor specific T regulatory cells. In summary, our current view from studies in the murine model is that the level of engraftment is limited by host cell competition, whereas loss of engraftment and absence of chimerism is because of failure to overcome the adaptive immune barrier.

Back to Article Outline

Considerations for Clinical Application of IUHCT 

There have been approximately 50 reported cases of IUHCT in humans over the past 20 years. Drawing conclusions based on clinical experience in humans has been difficult because of a large variety of target diseases, donor cell sources, and transplantation protocols. Not surprisingly, successes have largely been limited to cases of immunodeficiency syndromes in which donor cells have a clear selective advantage over host cells. In utero therapy for XSCID has been successful, with at least 10 documented cases of cellular reconstitution with functional T cells 65, 66, 67, 68, 82, 83. However, recipients manifest split chimerism with only the T cell compartment engrafted similar to the results of nonmyeloablative postnatal HSCT [84]. Thus far, there is no proved advantage for prenatal treatment of XSCID over neonatal transplantation. Attempts to treat other immunodeficiency disorders such as chronic granulomatous disease (CGD) or Chediak-Higashi syndrome, have been unsuccessful thus far, as all subjects were born without detectable engraftment 85, 86, 87, 88.

The use of IUHCT for hemoglobinopathies has also been attempted, but has thus far been largely unsuccessful. There have been 12 attempts to treat β-thalassemia in utero, with only 2 investigators reporting detectable postnatal engraftment 89, 90, 91, 92, 93, 94, at least 1 of whom subsequently lost engraftment [66]. There have been 3 reported attempts to treat α-thalassemia by IUHCT 90, 91, 92, 93, 94, 95, 96, with 1 patient exhibiting microchimerism and tolerance to donor antigen by mixed lymphocyte reaction [95]; however, all 3 patients remained transfusion dependent. There have also been 3 reported attempts to treat sickle cell anemia; however, none have resulted in detectable engraftment 88, 96. There have been 7 reported attempts to treat metabolic storage diseases by IUHCT 85, 91, 97, 98, with 2 reports of engraftment 82, 97, 1 of which led to no clinical improvement and the other resulted in prenatal death, likely because of GVHD.

Given this history, few recent attempts at IUHCT have been reported, and many investigators have been discouraged. However, the rationale remains compelling, and there are lessons to be learned from this experience that may help guide future efforts. Many of the historic attempts were ill advised for reasons that are now recognized. Many of the transplants were performed too late in gestation, or with donor cell sources that would not be expected to succeed. For instance, the use of highly enriched HSC as a donor source has been unsuccessful in allogeneic experimental systems, and has been clinically unsuccessful, even when performed under optimal circumstances. In addition, the expectation that one could achieve therapeutic levels of engraftment after IUHCT alone for diseases like the hemoglobinopathies was somewhat naïve given what we now understand about the barriers to engraftment.

At the present time, there are 2 clinical strategies that may be successful in clinical application. The first is IUHCT alone, which may be successful for selected biologically favorable target disorders. The second is IUHCT for donor-specific tolerance induction followed by postnatal minimally conditioned HSCT from the same donor. The latter approach holds the most immediate promise for broad clinical application of IUHCT because it requires only a minimal level of chimerism to be successful, and because it would be applicable to the majority of disorders that can be prenatally diagnosed and treated by postnatal HSCT.

Considerations for IUHCT Alone 

The goal of this strategy is to achieve therapeutic levels of engraftment with either single or multiple in utero transplants. Based on our current understanding of human immune and hematopoietic ontogeny, the ideal timing of at least the first IUHCT would be at 11 to 14 weeks gestation. During this time the fetal liver has active hematopoiesis, and thymic selection is ongoing with very few mature lymphocytes present in the thymus or peripheral circulation. Also, at this time the fetus is very small, <35 g in weight, allowing the opportunity to maximize the dose of donor cells. At the present time, the only disorders that this strategy can be contemplated for are disorders that offer either a competitive advantage for donor cells, or perhaps disorders that require only minimal levels of engraftment for therapeutic success. Clearly, the most biologically favorable disease for treatment by IUHCT alone remains XSCID. Other characterized mutations in cytokine receptor signaling pathways (ie, Jak 3 or ZAP-70) resulting in SCID should also be favorable candidate diseases for IUHCT. Based on the available clinical and experimental evidence, it is likely that any member of this group of disorders can be effectively treated by IUHCT, using established protocols, with results comparable to the reported results for XSCID. Neonatal nonmyeloablative haploidentical HSCT remains the standard for comparison for novel treatments of XSCID. For IUHCT to be recommended as an alternative therapy, a clear advantage for IUHCT would need to be demonstrated. Ideally, clinical trials of IUHCT for XSCID would be established, and the results compared to early postnatal transplantation protocols, to determine whether there is a biologic advantage favoring IUHCT. Unfortunately, such trials may not be possible because of the rarity of these diseases and the perception by some that postnatal therapy is adequate 84, 99. Another group of diseases that could benefit from IUHCT are those in which somatic mosaicism and in vivo selection have been documented to occur. In these diseases there is presumably a survival advantage for the spontaneously corrected cells [100]. Such correction has been noted in adenosine deaminase SCID [101], Fanconi Anemia [102], and Bloom Syndrome [103], the latter 2 of which are chromosomal breakage syndromes. In both Fanconi Anemia and Bloom Syndrome mitotic recombination was documented as the molecular mechanism of somatic reversion. This represents an experiment of nature documenting the improvement in a disease by clonal expansion of a single spontaneously corrected HSC, and suggests that low-level engraftment achieved in utero could eventually replace host hematopoiesis as progressive BM failure occurred. True clinical cure of either disease is unlikely, as they are associated with other pleiotropic manifestations, such as an increased rate of malignancy that are unlikely to be reversed by hematopoietic reconstitution alone.

Some diseases that are treatable with low levels of chimerism include CGD, hyper IgM syndrome, and leukocyte adhesion deficiency (LAD). It has been well documented that CGD can be corrected by as few as 5% normal neutrophils [104], and in X-linked hyper IgM syndrome, phenotypically normal carriers have been identified in whom the normal gene has been predominately silenced [105]. LAD-1 results from mutations in the leukocyte integrin CD18, which inhibits the expression of the CD18/CD11 complex on the cell surface and thus the ability of leukocytes to adhere to the vessel wall and migrate to sites of infection 106, 107, 108, 109. Recent studies in the analogous canine LAD model (CLAD) have demonstrated that even low levels of donor CD18+ cell engraftment following nonmyeloablative matched littermate BMT can reverse the lethal disease phenotype in CLAD 107, 110, 111, 112. We have recently demonstrated correction of the CLAD phenotype by IUHCT of haploidentical adult BM derived cells in the canine model [113].

Specific nonhematopoietic disorders of bone metabolism may also be attractive target disorders for IUHCT. A recent report of rescue of osteopetrotic mice with the same mutation as approximately half of human patients with the autosomal recessive diseases by IUHCT is intriguing [114]. In this study, complete phenotypic correction associated with osteoclast engraftment was achieved, despite the fact that an abundance of host osteoclasts remained present that were not functional. There is also interest in treatment of Osteogenesis Imperfecta by prenatal replacement of “mesenchymal stem cells” (MSCs) or stromal progenitor cells and a clinical case has been reported [115]. The experimental basis for application of IUHCT toward this disease, however, needs further development. Engraftment of MSCs after intraperitoneal transplantation in xenogeneic systems occurs but is very low in frequency [116]. The premise that typical MSCs isolated by plastic adherence can permanently repopulate the osteoblast compartment has been challenged [117] by the observation that hematopoietic cells and osteoblasts are derived from a common progenitor in the non-plastic adherent fraction of BM. The question is then whether IUHCT with BM cells can engraft the osteoblast compartment.

IUHCT alone could potentially treat the above disorders using currently available methodology and optimized protocols. However, at best, low levels of multilineage chimerism could be achieved. Ideally, approaches to achieve more robust engraftment by IUHCT will be developed (as discussed below). Until that time, however, the most likely strategy in the near future for IUHCT to treat many disorders is donor-specific tolerance induction followed by postnatal minimal conditioning HSCT.

IUHCT for Donor-Specific Tolerance Induction followed by Postnatal Minimal Conditioning HSCT 

As discussed earlier, low levels of mixed hematopoietic chimerism after IUHCT are associated with donor specific tolerance. The exact level of chimerism required may vary slightly with species, but is in the 1%-2% range. In the murine system, we have observed that in the presence of microchimerism (<0.5%, donor cells detectable only by PCR) approximately 1/3 of animals are tolerant of donor skin grafts and nonreactive by mixed lymphocyte reaction (MLR) 38, 42. Using the ability to enhance chimerism after IUHCT by a minimal conditioning postnatal HSCT from the same donor strain as the definition of tolerance, only 60% of animals with flow cytometrically detectable chimerism of <1% were tolerant, whereas 100% of animals with chimerism of >1% were tolerant [45]. Tolerance can be used as a platform to dramatically enhance donor cell chimerism postnatally by nontoxic approaches. We have demonstrated 3 such approaches in the murine model, that is, low-dose total body irradiation (TBI) [44], donor lymphocyte infusion (DLI) [43], and single-agent busulfan conditioning [45], all of which can increase donor chimerism to complete or near complete levels with minimal or no toxicity. This type of strategy is particularly well suited to the hemoglobinopathies [40], where moderate levels of mixed chimerism result in near complete replacement of circulating red cells because of the relatively prolonged half-life of normal red cells relative to diseased red cells. In the large Italian experience with mixed chimerism in β-thalassemia patients, levels of bone marrow chimerism of 25% have been associated with clinical amelioration of -thalassemia major and isolated observations support similar levels for sickle cell disease 118, 119. Currently, patients with sickle cell disease and Thalassemia are rarely transplanted in the absence of a matched sibling donor because of concern for treatment related toxicity. The ability to induce tolerance by IUHCT to a haploidentical parent would essentially create a matched donor circumstance, allowing a relatively nontoxic HSCT in the neonatal period.

Obviously, if hemoglobinopathies can be treated, many other disorders, including all of the disorders discussed above in which a selective advantage exists for donor cells or in which only minimal levels of engraftment are required for correction could also be treated. At the present time it is likely that, with optimal protocols, levels of chimerism required for tolerance induction can be achieved in human fetuses. What are currently needed are studies in preclinical large animal models that more closely approximate human fetal biology to optimize transplant regimens prior to human trials.

Back to Article Outline

Strategies to Improve Engraftment 

Consideration of IUHCT in the context of the barriers discussed above suggests a number of strategies by which higher levels of engraftment might be achieved. These strategies fall into 3 broad categories with significant overlap between them, which are: (1) strategies to improve the receptivity of the host and increase the number of donor HSC engrafted; (2) strategies to provide a competitive advantage for donor cells allowing expansion of donor cells after engraftment; and (3) strategies to overcome the immune barrier and, therefore, enhance the frequency of successful engraftment.

Improvement in host receptivity awaits the development of a method for highly selective, nontoxic myeloablation in the fetus. This is, in our opinion, the single development that would dramatically increase levels of donor cell engraftment and make IUHCT alone broadly applicable. The ability to selectively myeloablate the host hematopoietic compartment would allow donor cells to engraft and compete effectively after transplantation. Less dramatic improvement in receptivity might be achieved by nontoxic mobilization of host cells into the circulation so that hematopoietic niches would be available for donor HSC to occupy. Although theoretically attractive and powerful, there have been no studies published thus far in which either of these strategies have been successfully applied in the fetus.

Achieving a competitive advantage for donor cells is a promising strategy that is unlikely to achieve therapeutic levels of engraftment after IUHCT alone, but is very likely to be the key to achieving the incremental increase in engraftment needed for consistent and stable donor-specific tolerance induction. The simplest manipulation is to increase or maximize the number of donor cells transplanted. This can be approached by increasing the number of HSC within the donor innoculum, performing multiple transplants, or both. Similar to observations in the nonmyeloablated syngeneic mouse model discussed previously, both increases in cell dose and multiple transplants have been shown experimentally to incrementally increase engraftment after IUHCT [120]. Ex vivo manipulation of donor cells to selectively improve their competitive capacity after transplantation is an attractive strategy because it can be done prior to IUHCT, provides inherent selectivity for donor cells, and should avoid toxicity to the fetus. For instance, we recently demonstrated that blockade of the dipeptidyl peptidase CD26 [121] on donor cells increased homing of donor cells to the fetal liver, improved their competitive capacity relative to nonblocked cells, and resulted in increased short- and long-term levels of chimerism, presumably via a mechanism of increased SDF-1α expression on donor cells [51]. The demonstration that transient manipulation of a single chemokine interaction can result in improvements in engraftment is encouraging, as there are many steps in the homing and engraftment process that could potentially be manipulated, singly or in combination, to significantly improve engraftment. Other manipulations that selectively influence expression of homing receptors and engraftment include preincubation of donor cells with hematopoietic growth factors [122] and likely cotransplantation with stroma. The use of stromal cotransplantation has increased short- and long-term donor cell expression in the sheep model 123, 124; however, the mechanism remains to be elucidated.

The recognition of an immune barrier suggests potential immune-based strategies for application to IUHCT that might prevent loss of engraftment. However, intelligent design of such strategies awaits better definition of the nature and mechanism of the immune barrier. Historically, there have been a number of studies documenting the important role of donor T cells in engraftment. The concept that immune cells within the donor inoculum might facilitate engraftment is supported by observations of failure of engraftment of T cell-depleted or highly enriched HSC in multiple animal species and restoration of low level engraftment by the addition of T cells 30, 125, 126, 127. However, no dose of T cells has been identified that can meaningfully increase engraftment without the associated risk of GVHD. The strategy of inducing a graft-versus-hematopoietic effect by adding T cells from donors immunized against the recipient and treated ex vivo to reduce the likelihood of GVHD has been attempted in 2 studies using different techniques for T cell preparation 39, 128. In both studies proof of principle was demonstrated by the achievement of complete donor hematopoietic chimerism in selected animals. However, there was clear evidence of GVHD in both studies. The importance of these studies is that they demonstrate that if a more specific targeting strategy for host hematopoietic cells could be achieved by donor immune cells, very high levels of donor engraftment are possible. Such specificity might be possible, for example, by targeting hematopoietic specific minor histocompatibility antigens [129]. Finally, if graft rejection represents either failure of adequate thymic donor reactive lymphocyte deletion, and/or failure of donor specific T regulatory cell generation in the thymus, strategies related to improving the quantity and quality of donor antigen presentation in the thymus might be appropriate. Tipping the balance toward more complete donor reactive lymphocyte deletion or more robust generation of donor specific T regulatory cell generation may prevent rejection and allow consistent and reliable engraftment at levels that are adequate for donor specific tolerance induction.

Back to Article Outline

References 

  1. Billingham R, Brent L, Medawar PB. Actively acquired tolerance of foreign cells. Nature. 1953;172:603–607
  2. Palmer E. Negative selection—clearing out the bad apples from the T-cell repertoire. Nat Rev Immunol. 2003;3:383–391
  3. Sprent J. Central tolerance of T cells. Int Rev Immunol. 1995;13:95–105
  4. Takahama Y. Journey through the thymus: stromal guides for T-cell development and selection. Nat Rev Immunol. 2006;6:127–135
  5. Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol. 1995;155:1151–1164
  6. Takahashi T, Tagami T, Yamazaki S, et al. Immunologic self-tolerance maintained by CD25(+)CD4(+) regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4. J Exp Med. 2000;192:303–310
  7. Ziegler SF. FOXP3: of mice and men. Annu Rev Immunol. 2006;24:209–226
  8. Christensen JL, Wright DE, Wagers AJ, Weissman IL. Circulation and chemotaxis of fetal hematopoietic stem cells. PLoS Biol. 2004;2:E75
  9. Medvinsky A, Dzierzak E. Definitive hematopoiesis is autonomously initiated by the AGM region. Cell. 1996;86:897–906
  10. Mikkola HK, Gekas C, Orkin SH, Dieterlen-Lievre F. Placenta as a site for hematopoietic stem cell development. Exp Hematol. 2005;33:1048–1054
  11. Gill T. Chimerism in humans. Transplant Proc. 1977;9:1423–1431
  12. Jolly RD, Thompson KG, Murphy CE, Manktelow BW, Bruere AN, Winchester BG. Enzyme replacement therapy—an experiment of nature in a chimeric mannosidosis calf. Pediatr Res. 1976;10:219–224
  13. Owen RD. Immunogenetic consequences of vascular anastomoses between bovine cattle twins. Science. 1945;102:400–401
  14. Picus J, Aldrich WR, Letvin NL. A naturally occurring bone-marrow-chimeric primate. I. Integrity of its immune system. Transplantation. 1985;39:297–303
  15. Cragle R, Stone W. Preliminary results of kidney grafts between cattle chimeric twins. Transplantation. 1967;5:328–335
  16. Emery D, McCullagh P. Immunological reactivity between chimeric cattle twins. III. Mixed leukocyte reaction. Transplantation. 1980;29:17–22
  17. van Dijk B, Bommsma D, de Man A. Blood group chimerism in human multiple births is not rare. Am J Med Genet. 1996;61:264–268
  18. Picus J, Holley K, Aldrich WR, Griffin JD, Letvin NL. A naturally occurring bone marrow-chimeric primate. II. Environment dictates restriction on cytolytic T lymphocyte-target cell interactions. J Exp Med. 1985;162:2035–2052
  19. Hansen HE, Niebuhr E, Lomas C. Chimeric twins. T.S. and M.R. reexamined. Hum Hered. 1984;34:127–130
  20. Fleischman R, Mintz B. Prevention of genetic anemias in mice by microinjection of normal hematopoietic cells into the fetal placenta. Proc Natl Acad Sci USA. 1979;76:5736–5740
  21. Blazar BR, Taylor PA, Vallera DA. Adult bone marrow-derived pluripotent hematopoietic stem cells are engraftable when transferred in utero into moderately anemic fetal recipients. Blood. 1995;85:833–841
  22. Blazar BR, Taylor PA, Vallera DA. In utero transfer of adult bone marrow cells into recipients with severe combined immunodeficiency disorder yields lymphoid progeny with T- and B-cell functional capabilities. Blood. 1995;86:4353–4366
  23. Blazer BR, Taylor PA, McElmurry R, et al. Engraftment of severe combined immune deficient mice receiving allogeneic bone marrow via in utero or postnatal transfer. Blood. 1998;92:3949–3959
  24. Shultz LD, Schweitzer PA, Christianson SW, et al. Multiple defects in innate and adaptive immunologic function in NOD/LtSz-scid mice. J Immunol. 1995;154:180–191
  25. Archer DR, Turner CW, Yeager AM, Fleming WH. Sustained multilineage engraftment of allogeneic hematopoietic stem cells in NOD/SCID mice after in utero transplantation. Blood. 1997;90:3222–3229
  26. Flake AW, Harrison MR, Adzick NS, Zanjani ED. Transplantation of fetal hematopoietic stem cells in utero: the creation of hematopoietic chimeras. Science. 1986;233:776–778
  27. Zanjani ED, Ascensao JL, Flake AW, Harrison MR, Tavassoli M. The fetus as an optimal donor and recipient of hemopoietic stem cells. Bone Marrow Transplant. 1992;10(Suppl 1):107–114
  28. Zanjani ED, Flake AW, Rice H, Hedrick M, Tavassoli M. Long-term repopulating ability of xenogeneic transplanted human fetal liver hematopoietic stem cells in sheep. J Clin Invest. 1994;93:1051–1055
  29. Zanjani ED, Pallavicini MG, Flake AW, et al. Engraftment and long-term expression of human fetal hemopoietic stem cells in sheep following transplantation in utero. J Clin Invest. 1992;89:1178–1188
  30. Lee PW, Cina RA, Randolph MA, et al. Stable multilineage chimerism across full MHC barriers without graft-versus-host disease following in utero bone marrow transplantation in pigs. Exp Hematol. 2005;33:371–379
  31. Lee PW, Cina RA, Randolph MA, et al. In utero bone marrow transplantation induces kidney allograft tolerance across a full major histocompatibility complex barrier in Swine. Transplantation. 2005;79:1084–1090
  32. Harrison MR, Slotnick RN, Crombleholme TM, Golbus MS, Tarantal AF, Zanjani ED. In-utero transplantation of fetal liver haemopoietic stem cells in monkeys. Lancet. 1989;2:1425–1427
  33. Shields LE, Bryant EM, Easterling TR, Andrews RG. Fetal liver cell transplantation for the creation of lymphohematopoietic chimerism in fetal baboons. Am J Obstet Gynecol. 1995;173:1157–1160
  34. Pearce R, Kiehm D, Armstrong D, et al. Induction of hematopoietic chimerism in the caprine fetus by intraperitoneal injection of fetal liver cells. Experientia. 1989;45:307–308
  35. Blakemore K, Hattenburg C, Stetten G, et al. In utero hematopoietic stem cell transplantation with haploidentical donor adult bone marrow in a canine model. Am J Obstet Gynecol. 2004;190:960–973
  36. Carrier E, Lee TH, Busch MP, Cowan MJ. Induction of tolerance in nondefective mice after in utero transplantation of major histocompatibility complex-mismatched fetal hematopoietic stem cells. Blood. 1995;86:4681–4690
  37. Donahue J, Gilpin E, Lee TH, Busch MP, Croft M, Carrier E. Microchimerism does not induce tolerance and sustains immunity after in utero transplantation. Transplantation. 2001;71:359–368
  38. Kim HB, Shaaban AF, Yang EY, Liechty KW, Flake AW. Microchimerism and tolerance after in utero bone marrow transplantation in mice. J Surg Res. 1998;77:1–5
  39. Hayashi S, Hsieh M, Peranteau WH, Ashizuka S, Flake AW. Complete allogeneic hematopoietic chimerism achieved by in utero hematopoietic cell transplantation and cotransplantation of LLME-treated, MHC-sensitized donor lymphocytes. Exp Hematol. 2004;32:290–299
  40. Hayashi S, Abdulmalik O, Peranteau WH, et al. Mixed chimerism following in utero hematopoietic stem cell transplantation in murine models of hemoglobinopathy. Exp Hematol. 2003;31:176–184
  41. Shaaban AF, Kim HB, Milner R, Flake AW. A kinetic model for the homing and migration of prenatally transplanted marrow. Blood. 1999;94:3251–3257
  42. Kim HB, Shaaban AF, Milner R, Fichter C, Flake AW. In utero bone marrow transplantation induces tolerance by a combination of clonal deletion and anergy. J Pediatr Surg. 1999;34:726–730
  43. Hayashi S, Peranteau WH, Shaaban AF, Flake AW. Complete allogeneic hematopoietic chimerism achieved by a combined strategy of in utero hematopoietic stem cell transplantation and postnatal donor lymphocyte infusion. Blood. 2002;100:804–812
  44. Peranteau WF, Hayashi S, Hsieh M, Shaaban AF, Flake AW. High level allogeneic chimerism achieved by prenatal tolerance induction and postnatal non-myeloablative bone marrow transplantation. Blood. 2002;100:2225–2234
  45. Ashizuka S, Peranteau WH, Hayashi S, Flake AW. Busulfan-conditioned bone marrow transplantation results in high-level allogeneic chimerism in mice made tolerant by in utero hematopoietic cell transplantation. Exp Hematol. 2006;34:359–368
  46. Flake AW, Zanjani ED. In utero hematopoietic stem cell transplantation: ontogenic opportunities and biologic barriers. Blood. 1999;94:2179–2191
  47. Rao SS, Peters SO, Crittenden RB, Stewart FM, Ramshaw HS, Quesenberry PJ. Stem cell transplantation in the normal nonmyeloablated host: relationship between cell dose, schedule, and engraftment. Exp Hematol. 1997;25:114–121
  48. Stewart FM, Crittenden RB, Lowry PA, Pearson-White S, Quesenberry PJ. Long-term engraftment of normal and post-5-fluorouracil murine marrow into normal nonmyeloablated mice. Blood. 1993;81:2566–2571
  49. Ramshaw HS, Crittenden RB, Dooner M, Peters SO, Rao SS, Quesenberry PJ. High levels of engraftment with a single infusion of bone marrow cells into normal unprepared mice. Biol Blood Marrow Transplant. 1995;1:74–80
  50. Peranteau WH, Endo M, Adibe OO, Flake AW. Evidence for an immune barrier after in utero hematopoietic-cell transplantation. Blood. 2007;109:1331–1333
  51. Peranteau WH, Endo M, Adibe OO, Merchant A, Zoltick PW, Flake AW. CD26 inhibition enhances allogeneic donor-cell homing and engraftment after in utero hematopoietic-cell transplantation. Blood. 2006;108:4268–4274
  52. Wolf NS, Bertoncello I, Jiang D, Priestley G. Developmental hematopoiesis from prenatal to young-adult life in the mouse model. Exp Hematol. 1995;23:142–146
  53. Harrison DE, Astle CM. Short- and long-term multilineage repopulating hematopoietic stem cells in late fetal and newborn mice: models for human umbilical cord blood transplantation. Blood. 1997;90:174–181
  54. Mintz B, Anthony K, Litwin S. Monoclonal derivation of mouse myeloid and lymphoid lineages from totipotent hematopoietic stem cells experimentally engrafted in fetal hosts. Proc Natl Acad Sci USA. 1984;81:7835–7839
  55. Harrison DE, Zhong RK, Jordan CT, Lemischka IR, Astle CM. Relative to adult marrow, fetal liver repopulates nearly 5 times more effectively long-term than short-term. Exp Hematol. 1997;25:293–297
  56. Jordan CT, Astle CM, Zawadzki J, Mackarehtschian K, Lemischka IR, Harrison DE. Long-term repopulating abilities of enriched fetal liver stem cells measured by competitive repopulation. Exp Hematol. 1995;23:1011–1015
  57. Rebel VI, Miller CL, Eaves CJ, Lansdorp PM. The repopulating potential of fetal liver hematopoietic stem cells in mice exceeds that of their liver adult bone marrow counterparts. Blood. 1996;87:3500–3507
  58. Rebel VI, Miller CL, Thornbury GR, Dragowska WH, Eaves CJ, Lansdorp PM. A comparison of long-term repopulating hematopoietic stem cells in fetal liver and adult bone marrow from the mouse. Exp Hematol. 1996;24:638–648
  59. Leung W, Ramirez M, Civin CI. Quantity and quality of engrafting cells in cord blood and autologous mobilized peripheral blood. Biol Blood Marrow Transplant. 1999;5:69–76
  60. Rosler ES, Brandt JE, Chute J, Hoffman R. An in vivo competitive repopulation assay for various sources of human hematopoietic stem cells. Blood. 2000;96:3414–3421
  61. Van Zant G, Chen JJ, Scott-Micus K. Developmental potential of hematopoietic stem cells determined using retrovirally marked allophenic marrow. Blood. 1991;77:756–763
  62. Van Zant G, Holland BP, Eldridge PW, Chen JJ. Genotype-restricted growth and aging patterns in hematopoietic stem cell populations of allophenic mice. J Exp Med. 1990;171:1547–1565
  63. Mintz B, Palm J. Gene control of hematopoiesis. I. Erythrocyte mosaicism and permanent immunological tolerance in allophenic mice. J Exp Med. 1969;129:1013–1027
  64. Stewart FM, Zhong S, Wuu J, Hsieh C-c, Nilsson SK, Quesenberry PJ. Lymphohematopoietic engraftment in minimally myeloablated hosts. Blood. 1998;91:3681–3687
  65. Flake A, Roncarolo M-G, Puck J, et al. Treatment of X-linked severe combined immunodeficiency by in utero transplantation of paternal bone marrow. N Engl J Med. 1996;335:1806–1810
  66. Touraine JL, Raudrant D, Rebaud A, et al. In utero transplantation of stem cells in humans: immunological aspects and clinical follow-up of patients. Bone Marrow Transplant. 1992;1:121–126
  67. Touraine JL, Raudrant D, Royo C, et al. In-utero transplantation of stem cells in bare lymphocyte syndrome. Lancet. 1989;1:1382
  68. Westgren M, Ringden O, Bartmann P, et al. Prenatal T-cell reconstitution after in utero transplantation with fetal liver cells in a patient with X-linked severe combined immunodeficiency. Am J Obstet Gynecol. 2002;187:475–482
  69. Renda MC, Fecarotta E, Dieli F, et al. Evidence of alloreactive T lymphocytes in fetal liver: implications for fetal hematopoietic stem cell transplantation. Bone Marrow Transplant. 2000;25:135–141
  70. Lindton B, Markling L, Ringden O, Kjaeldgaard A, Gustafson O, Westgren M. Mixed lymphocyte culture of human fetal liver cells. Fetal Diagn Ther. 2000;15:71–78
  71. Toivanen P, Uksila J, Leino A, Lassila O, Hirvonen T, Ruuskanen O. Development of mitogen responding T cells and natural killer cells in the human fetus. Immunol Rev. 1981;57:89–105
  72. Lindton B, Markling L, Ringden O, Westgren M. In vitro studies of the role of CD3+ and CD56+ cells in fetal liver cell alloreactivity. Transplantation. 2003;76:204–209
  73. Gotherstrom C, Johnsson AM, Mattsson J, Papadogiannakis N, Westgren M. Identification of maternal hematopoietic cells in a 2nd-trimester fetus. Fetal Diagn Ther. 2005;20:355–358
  74. Setiady YY, Samy ET, Tung KS. Maternal autoantibody triggers de novo T cell-mediated neonatal autoimmune disease. J Immunol. 2003;170:4656–4664
  75. Shields LE, Gaur L, Delio P, Potter J, Sieverkropp A, Andrews RG. Fetal immune suppression as adjunctive therapy for in utero hematopoietic stem cell transplantation in nonhuman primates. Stem Cells. 2004;22:759–769
  76. Fleischman RA, Mintz B. Development of adult bone marrow stem cells in H-2 compatable and noncompatable mouse fetuses. J Exp Med. 1984;159:159–169
  77. Howson-Jan K, Matloub YH, Vallera DA, Blazar BR. In utero engraftment of fully H-2-incompatible versus congenic adult bone marrow transferred into nonanemic or anemic murine fetal recipients. Transplantation. 1993;56:709–716
  78. Barker JE, Schuldt AJ, Lessard MD, Jude CD, Vogler CA, Soper BW. Donor cell replacement in mice transplanted in utero is limited by immune-independent mechanisms. Blood Cells Mol Dis. 2003;3:291–297
  79. Blackman M, Kappler J, Marrack P. The role of the T-cell receptor in positive and negative selection of developing T-cells. Science. 1990;248:1335–1342
  80. Schwartz R. Acquisition of immunologic self tolerance. Cell. 1989;57:1073–1081
  81. Sha WC, Nelson CA, et al. Positive and negative selection of an antigen receptor on T-cells in transgenic mice. Nature. 1988;336:73–76
  82. Touraine JL, Raudrant D, Golfier F, et al. Reappraisal of in utero stem cell transplantation based on long-term results. Fetal Diagn Ther. 2004;19:305–312
  83. Wengler G, Lanfranchi A, Frusca T, et al. In-utero transplantation of parental CD34 haematopoietic progenitor cells in a patient with X-linked severe combined immunodeficiency (SCIDX1). Lancet. 1996;348:1484–1487
  84. Buckley RH, Schiff SE, Schiff RI, et al. Hematopoietic stem-cell transplantation for the treatment of severe combined immunodeficiency. N Engl J Med. 1999;340:508–516
  85. Flake AW, Zanjani ED. In utero hematopoietic stem cell transplantation. A status report. JAMA. 1997;278:932–937
  86. Jones DR, Bui TH, Anderson EM, et al. In utero haematopoietic stem cell transplantation: current perspectives and future potential. Bone Marrow Transplant. 1996;18:831–837
  87. Muench MO, Rae J, Barcena A, et al. Transplantation of a fetus with paternal Thy-1(+)CD34(+)cells for chronic granulomatous disease. Bone Marrow Transplant. 2001;27:355–364
  88. Westgren M. In utero stem cell transplantation. Semin Reprod Med. 2006;24:348–357
  89. Cowan M, Golbus M. In utero hematopoietic stem cell transplants for inherited disease. Am J Pediatr Hematol Oncol. 1994;16:35–42
  90. Diukman R, Golbus MS. In utero stem cell therapy. J Reprod Med. 1992;37:515–520
  91. Slavin S, Naparstek E, Ziegler M, et al. Clinical application of intrauterine bone marrow transplantation for treatment of genetic disease—feasibility studies. Bone Marrow Transplant. 1992;1:189–190
  92. Touraine J. Treatment of human fetuses and induction of immunological tolerance in humans by in utero transplantation of stem cells into fetal recipients. Acta Haematol. 1996;96:115–119
  93. Orlandi F, Giambona A, Messana F, et al. Evidence of induced non-tolerance in HLA-identical twins with hemoglobinopathy after in utero fetal transplantation. Bone Marrow Transplant. 1996;18:637–639
  94. Monni G, Ibba RM, Zoppi MA, Floris M. In utero stem cell transplantation. Croat Med J. 1998;39:220–223
  95. Hayward A, Ambruso D, Battaglia F, et al. Microchimerism and tolerance following intrauterine transplantation and transfusion for a-thalassemia-1. Fetal Diagn Ther. 1998;13:8–14
  96. Westgren M, Ringden O, Sturla E-N, et al. Lack of evidence of permanent engraftment after in utero fetal stem cell transplantation in congenital hemoglobinopathies. Transplantation. 1996;61:1176–1179
  97. Bambach BJ, Moser HW, Blakemore K, et al. Engraftment following in utero bone marrow transplantation for globoid cell leukodystrophy. Bone Marrow Transplant. 1997;19:399–402
  98. Touraine JL, Raudrant D, Laplace S. Transplantation of hemopoietic cells from the fetal liver to treat patients with congenital diseases postnatally or prenatally. Transplant Proc. 1997;29:712–713
  99. Flake AW, Zanjani ED. Treatment of severe combined immunodeficiency. Letter to the Editor. N Engl J Med. 1999;341:291
  100. Kvittingen EA, Rootwelt H, Brandtzaeg P, Bergan A, Berger R. Hereditary tyrosinemia type I. Self-induced correction of the fumarylacetoacetase defect. J Clin Invest. 1993;91:1816–1821
  101. Hirschhorn R, Yang DR, Puck JM, Huie ML, Jiang CK, Kurlandsky LE. Spontaneous in vivo reversion to normal of an inherited mutation in a patient with adenosine deaminase deficiency. Nat Genet. 1996;13:290–296
  102. D'Andrea AD, Grompe M. Molecular biology of Fanconi anemia: implications for diagnosis and therapy. Blood. 1997;90:1725–1736
  103. Ellis NA, Lennon DJ, Proytcheva M, Aldadeff B, Henderson EE, German J. Somatic intragenic recombination within the mutated locus BLM can correct the high sister-chromatid exchange phenotype of Bloom Syndrome cells. Am J Hum Genet. 1995;57:1019–1027
  104. Bjorgvinsdottir H, Ding C, Pech N, Gifford M, Li L, Dinauer M. Retroviral-mediated gene transfer of gp91phox into bone marrow cells rescues defect in host defense against Aspergillus fumigatus in murine X-linked chronic granulomatous disease. Blood. 1997;89:41–48
  105. Hollenbaugh D, Wu LH, Ochs HD, et al. The random inactivation of the X chromosome carrying the defective gene responsible for X-linked hyper IgM syndrome (X-HIM) in female carriers of HIGM1. J Clin Invest. 1994;94:616–622
  106. Anderson DC, Springer TA. Leukocyte adhesion deficiency: an inherited defect in the Mac-1, LFA-1, and p150,95 glycoproteins. Annu Rev Med. 1987;38:175–194
  107. Creevy KE, Bauer TR, Tuschong LM, et al. Canine leukocyte adhesion deficiency colony for investigation of novel hematopoietic therapies. Vet Immunol Immunopathol. 2003;94:11–22
  108. Kishimoto TK, Hollander N, Roberts TM, Anderson DC, Springer TA. Heterogeneous mutations in the beta subunit common to the LFA-1, Mac-1, and p150,95 glycoproteins cause leukocyte adhesion deficiency. Cell. 1987;50:193–202
  109. Springer TA, Thompson WS, Miller LJ, Schmalstieg FC, Anderson DC. Inherited deficiency of the Mac-1, LFA-1, p150,95 glycoprotein family and its molecular basis. J Exp Med. 1984;160:1901–1918
  110. Bauer TR, Creevy KE, Gu YC, et al. Very low levels of donor CD18+ neutrophils following allogeneic hematopoietic stem cell transplantation reverse the disease phenotype in canine leukocyte adhesion deficiency. Blood. 2004;103:3582–3589
  111. Bauer TR, Gu YC, Tuschong LM, et al. Nonmyeloablative hematopoietic stem cell transplantation corrects the disease phenotype in the canine model of leukocyte adhesion deficiency. Exp Hematol. 2005;33:706–712
  112. Sokolic RA, Bauer TR, Gu YC, et al. Nonmyeloablative conditioning with busulfan before matched littermate bone marrow transplantation results in reversal of the disease phenotype in canine leukocyte adhesion deficiency. Biol Blood Marrow Transplant. 2005;11:755–763
  113. Peranteau WH, Gu Y, Volk S, et al. In utero hematopoietic cell transplantation using haploidentical parental donors reverses the lethal phenotype in dogs with canine leukocyte adhesion deficiency. Blood. 2006;108:188a
  114. Frattini A, Blair HC, Sacco MG, et al. Rescue of ATPa3-deficient murine malignant osteopetrosis by hematopoietic stem cell transplantation in utero. Proc Natl Acad Sci USA. 2005;102:14629–14634
  115. Le Blanc K, Gotherstrom C, Ringden O, et al. Fetal mesenchymal stem-cell engraftment in bone after in utero transplantation in a patient with severe osteogenesis imperfecta. Transplantation. 2005;79:1607–1614
  116. Liechty KW, MacKenzie TC, Shaaban AF, et al. Human mesenchymal stem cells engraft and demonstrate site-specific differentiation after in utero transplantation in sheep. Nat Med. 2000;6:1282–1286
  117. Dominici M, Pritchard C, Garlits JE, Hofmann TJ, Persons DA, Horwitz EM. Hematopoietic cells and osteoblasts are derived from a common marrow progenitor after bone marrow transplantation. Proc Natl Acad Sci USA. 2004;101:11761–11766
  118. Andreani M, Nesci S, Lucarelli G, et al. Long-term survival of ex-thalassemic patients with persistent mixed chimerism after bone marrow transplantation. Bone Marrow Transplant. 2000;25:401–404
  119. Walters MC, Patience M, Leisenring W, et al. Bone marrow transplantation for sickle cell disease. [see comments] N Engl J Med. 1996;335:369–376
  120. Flake AW, Zanjani ED. In utero transplantation. In:  Bloom KG,  Forman SJ,  Applebaum FR editor. Thomas' Hematopoietic Cell Transplantation. 3rd ed.. Malden, MA: Blackwell Science Inc.; 2004;p. 565–575
  121. Christopherson KW, Hangoc G, Broxmeyer HE. Cell surface peptidase CD26/dipeptidylpeptidase IV regulates CXCL12/stromal cell-derived factor-1 alpha-mediated chemotaxis of human cord blood CD34+ progenitor cells. J Immunol. 2002;169:7000–7008
  122. Shaaban AF, Kim HB, Gaur L, Liechty KW, Flake AW. Prenatal transplantation of cytokine-stimulated marrow improves early chimerism in a resistant strain combination but results in poor long-term engraftment. Exp Hematol. 2006;34:1278–1287
  123. Almeida-Porada G, Flake AW, Glimp HA, Zanjani ED. Co-transplantation of stroma results in enhancement of engraftment and early expression of donor hematopoietic stem cell in utero. Exp Hematol. 1999;27:1569–1575
  124. Almeida-Porada G, Flake AW, Zanjani ED. Cotransplantation of sheep stroma results in enhancement of engraftment and early expression of donor hematopoietic stem cells in utero. Blood. 1998;92(Suppl 1):116a
  125. Crombleholme TM, Harrison MR, Zanjani ED. In utero transplantation of hematopoietic stem cells in sheep: the role of T cells in engraftment and graft-versus-host disease. J Pediatr Surg. 1990;25:885–892
  126. Shields LE, Gaur LK, Gough M, Potter J, Sieverkropp A, Andrews RG. In utero hematopoietic stem cell transplantation in nonhuman primates: the role of T cells. Stem Cells. 2003;21:304–314
  127. Petersen SM, Gendelman M, Murphy KM, et al. Use of T-cell antibodies for donor dosaging in a canine model of in utero hematopoietic stem cell transplantation. Fetal Diagn Ther. 2007;22:175–179
  128. Bhattacharyya S, Chawla A, Smith K, et al. Multilineage engraftment with minimal graft-versus-host disease following in utero transplantation of S-59 psoralen/ultraviolet a light-treated, sensitized T cells and adult T cell-depleted bone marrow in fetal mice. J Immunol. 2002;169:6133–6140
  129. den Haan JM, Meadows LM, Wang W, et al. The minor histocompatibility antigen HA-1: a diallelic gene with a single amino acid polymorphism. Human minor histocompatibility antigens: new concepts for marrow transplantation and adoptive immunotherapy. Science. 1998;279:1054–1057

 The authors have no potential conflicts of interest to disclose.

PII: S1083-8791(08)00089-X

doi:10.1016/j.bbmt.2008.02.012

Biology of Blood and Marrow Transplantation
Volume 14, Issue 7 , Pages 729-740, July 2008

Article Tools